mTOR-inhibitor-containing medicine for treating or preventing ophthalmic symptoms, disorders, or diseases, and application thereof

ABSTRACT

The present invention provides a medicine and a method for preventing or treating ophthalmic symptoms, disorders, or diseases. The present invention provides an mTOR-inhibitor-containing composition for preventing or treating ophthalmic symptoms, disorders, or diseases. In some of the embodiments of the present invention, this composition is capable of treating or preventing corneal endothelial symptoms, disorders, or diseases; in particular, corneal endothelial symptoms, disorders, or diseases that are attributed to overexpression of the transforming growth factor-β (TGF-β) signal and/or extracellular matrix (ECM).

TECHNICAL FIELD

The present invention relates to a medicament for treating or preventingeye conditions, disorders, or diseases, comprising an mTOR (alsoreferred to as mechanistic target of rapamycin mammalian target ofrapamycin) inhibitor and application thereof.

BACKGROUND ART

Visual information is recognized when light transmitted into the cornea,which is a transparent tissue at the front-most part of an eye ball,reaches the retina and excites nerve cells of the retina, and agenerated electrical signal is transmitted through the optic nerve tothe visual cortex of the cerebrum. To attain good vision, it isnecessary that the cornea is transparent. The transparency of the corneais retained by maintaining constant water content with pumping andbarrier functions of corneal endothelial cells.

Human corneal endothelial cells are present at a density of about 3000cells per 1 mm² at birth. Once damaged, human corneal endothelial cellshave a very limited ability to regenerate. Fuchs' endothelial cornealdystrophy is a disease that causes abnormality in endothelial cellsinside the cornea and significantly reduces the density of cornealendothelial cells, resulting in edema of the cornea. The cause thereofis unknown. In Fuchs' endothelial corneal dystrophy, extracellularmatrix such as collagen or fibronectin is deposited on a part of theback surface of a Descemet's membrane at the back of the cornea,resulting in guttae (Corneal guttae) and hypertrophy of the Descemet'smembrane. Guttae (Corneal guttae) and hypertrophy of the Descemet'smembrane are the cause of photophobia or blurred vision in Fuchs'endothelial corneal dystrophy patients, which significantly compromisesthe QOL of the patients. In this manner, extracellular matrix such asfibronectin is associated with conditions that cause reduced visualacuity such as guttata on the corneal endothelial surface or turbidguttae, and can be the main cause of a corneal endothelial disorderassociated with opacity of the cornea such as clouding, cornealturbidity or leucoma. It is understood that there is no effectivetherapeutic method other than corneal transplant for Fuchs' endothelialcorneal dystrophy. However, there is a shortage in cornea donation inJapan, where the number of patients waiting for corneal transplant isabout 2600, whereas the number of corneal transplants performed in Japanis approximately 1700 annually.

For Fuchs' endothelial corneal dystrophy, culture (Non PatentLiteratures 1 and 3) and immortalization (Non Patent Literature 2) ofcorneal endothelial cells from Fuchs' corneal dystrophy patients havebeen reported, but cells suitable for screening of a therapeutic drug orprogression preventing drug which maintain the features of the disease,such as overproduction of extracellular matrices, have not beenreported. Therefore, there is a limit to the development of atherapeutic drug thereof. Currently, there is no therapeutic drug thatis used in clinical practice, so that therapy is reliant on cornealtransplant.

CITATION LIST Non Patent Literature

-   [NPL 1] Zaniolo K, et al. Exp Eye Res.; 94 (1): 22-31. 2012-   [NPL 2] Azizi B, et al. Invest Ophthalmol Vis Sci. 2; 52 (13):    9291-9297. 2011-   [NPL 3] Kelliher C. et al. Exp Eye Res Vol. 93 (6), 880-888, 2011

SUMMARY OF INVENTION Solution to Problem

The inventors have discovered that inhibition of an mTOR suppresses celldeath (apoptosis) of an eye, especially corneal endothelial cells, andcan be applied to the use for treating or preventing ophthalmic diseasessuch as corneal endothelial disorders (especially corneal endothelialdisorders in Fuchs' endothelial corneal dystrophy) due to a transforminggrowth factor-β (TGF-β). In addition, the inventors have unexpectedlydiscovered that overexpression of extracellular matrix (ECM) such asfibronectin can be suppressed by inhibiting mTOR. The inventors havethereby discovered that an mTOR inhibitor can be applied to theimprovement, therapy, or prevention of corneal endothelial diseases dueto overexpression of extracellular matrix (e.g., guttae, hypertrophy ofthe Descemet's layer, corneal turbidity, leucoma, other conditions ofclouding, and the like). Since cell death and extracellular matrix areindependent events, it is preferable that both can be suppressed.

The present invention therefore provides, for example, the followingitems.

(Item 1)

A composition for use in preventing or treating an eye condition,disorder, or disease, comprising an mTOR inhibitor.

(Item 2)

The composition of item 1, wherein the eye condition, disorder, ordisease is a corneal endothelial condition, disorder, or disease.

(Item 3)

The composition of item 1 or 2, wherein the eye condition, disorder, ordisease is a corneal endothelial condition, disorder, or disease due toa transforming growth factor-β (TGF-β).

(Item 4)

The composition of any one of items 1 to 3, wherein the cornealendothelial condition, disorder, or disease is selected from the groupconsisting of Fuchs' endothelial corneal dystrophy, post-cornealtransplant disorder, corneal endotheliitis, trauma, ophthalmic surgery,post-ophthalmic laser surgery disorder, aging, posterior polymorphousdystrophy (PPD), congenital hereditary endothelial dystrophy (CHED),idiopathic corneal endothelial disorder, and cytomegalovirus cornealendotheliitis.

(Item 5)

The composition of any one of items 1 to 4, wherein the cornealendothelial condition, disorder, or disease is due to overexpression ofextracellular matrix (ECM).

(Item 6)

The composition of item 5, wherein the corneal endothelial condition,disorder, or disease is selected from the group consisting of Fuchs'endothelial corneal dystrophy, guttae formation, hypertrophy of aDescemet's membrane, hypertrophy of a cornea, turbidity, scar, cornealnebula, corneal macula, corneal leucoma, photophobia, and blurredvision.

(Item 7)

The composition of any one of items 1 to 6, wherein the condition,disorder, or disease comprises Fuchs' endothelial corneal dystrophy.

(Item 8)

The composition of any one of items 1 to 7, wherein the mTOR inhibitoris selected from the group consisting of rapamycin, temsirolimus,everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalisib,Ridaforolimus, NVP-BEZ235, CZ415, Torkinib, Torin 1, Omipalisib,OSI-027, PF-04691502, Apitolisib, WYE-354, Vistusertib, Torin 2,Tacrolimus, GSK1059615, Gedatolisib, WYE-125132, BGT226, Palomid 529,PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388,Zotarolimus, and Chrysophanic Acid.

(Item 9)

The composition of any one of items 1 to 7, wherein the mTOR inhibitoris an mTOR gene expression suppressing substance.

(Item 10)

The composition of item 9, wherein the mTOR gene expression suppressingsubstance is siRNA, antisense nucleic acid, or ribozyme against an mTORgene.

(Item 11)

The composition of item 9 or 10, wherein the mTOR gene expressionsuppressing substance is siRNA against an mTOR gene, wherein the siRNAcomprises a sense strand consisting of a nucleic acid sequence set forthin SEQ ID NO: 1 or the nucleic acid sequence wherein 1 to 3 bases ofnucleotides are deleted, substituted, inserted and/or added, and anantisense strand consisting of a nucleic acid sequence set forth in SEQID NO: 2 or the nucleic acid sequence wherein 1 to 3 bases ofnucleotides are deleted, substituted, inserted and/or added.

(Item 12)

The composition of any one of items 1 to 8, wherein the mTOR inhibitoris selected from the group consisting of rapamycin, temsirolimus, andeverolimus.

(Item 13)

The composition of any one of items 1 to 12, wherein the composition isan eye drop.

(Item 14)

The composition of any one of items 1 to 8, wherein the mTOR inhibitoris rapamycin and is present in the composition at at least about 0.1 nM.

(Item 15)

The composition of any one of items 1 to 8, wherein the composition isan eye drop, wherein the mTOR inhibitor is rapamycin and is present inthe eye drop at at least about 0.1 mM.

(Item 16)

The composition of any one of items 1 to 8, wherein the mTOR inhibitoris temsirolimus and is present in the composition at at least about 0.01nM.

(Item 17)

The composition of any one of items 1 to 8, wherein the composition isan eye drop, wherein the mTOR inhibitor is temsirolimus and is presentin the eye drop at at least about 0.01 mM.

(Item 18)

The composition of any one of items 1 to 8, wherein the mTOR inhibitoris everolimus and is present in the composition at at least about 0.1nM.

(Item 19)

The composition of any one of items 1 to 8, wherein the composition isan eye drop, wherein the mTOR inhibitor is everolimus and is present inthe eye drop at at least about 0.1 mM.

(Item 1A)

A method for preventing or treating an eye condition, disorder, ordisease in a subject, wherein the method comprises administering aneffective amount of an mTOR inhibitor to the subject.

(Item 2A)

The method of item 1A, wherein the eye condition, disorder, or diseaseis a corneal endothelial condition, disorder, or disease.

(Item 3A)

The method of item 1A or 2A, wherein the eye condition, disorder, ordisease is a corneal endothelial condition, disorder, or disease due toa transforming growth factor-β (TGF-β).

(Item 4A)

The method of any one of items 1A to 3A, wherein the corneal endothelialcondition, disorder, or disease is selected from the group consisting ofFuchs' endothelial corneal dystrophy, post-corneal transplant disorder,corneal endotheliitis, trauma, ophthalmic surgery, post-ophthalmic lasersurgery disorder, aging, posterior polymorphous dystrophy (PPD),congenital hereditary endothelial dystrophy (CHED), idiopathic cornealendothelial disorder, and cytomegalovirus corneal endotheliitis.

(Item 5A)

The method of any one of items 1A to 4A, wherein the corneal endothelialcondition, disorder, or disease is due to overexpression ofextracellular matrix (ECM).

(Item 6A)

The method of item 5A, wherein the corneal endothelial condition,disorder, or disease is selected from the group consisting of Fuchs'endothelial corneal dystrophy, guttae formation, hypertrophy of aDescemet's membrane, hypertrophy of a cornea, turbidity, scar, cornealnebula, corneal macula, leucoma, photophobia, and blurred vision.

(Item 7A)

The method of any one of items 1A to 6A, wherein the condition,disorder, or disease comprises Fuchs' endothelial corneal dystrophy.

(Item 8A)

The method of any one of items 1A to 7A, wherein the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus,everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalisib,Ridaforolimus, NVP-BEZ235, CZ415, Torkinib, Torin 1, Omipalisib,OSI-027, PF-04691502, Apitolisib, WYE-354, Vistusertib, Torin 2,Tacrolimus, GSK1059615, Gedatolisib, WYE-125132, BGT226, Palomid 529,PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388,Zotarolimus, and Chrysophanic Acid.

(Item 9A)

The method of any one of items 1A to 7A, wherein the mTOR inhibitor isan mTOR gene expression suppressing substance.

(Item 10A)

The method of item 9A, wherein the mTOR gene expression suppressingsubstance is siRNA, antisense nucleic acid, or ribozyme against an mTORgene.

(Item 11A)

The method of item 9A or 10A, wherein the mTOR gene expressionsuppressing substance is siRNA against an mTOR gene, wherein the siRNAcomprises a sense strand consisting of a nucleic acid sequence set forthin SEQ ID NO: 1 or the nucleic acid sequence wherein 1 to 3 bases ofnucleotides are deleted, substituted, inserted and/or added, and anantisense strand consisting of a nucleic acid sequence set forth in SEQID NO: 2 or a nucleic acid sequence wherein 1 to 3 bases of nucleotidesare deleted, substituted, inserted and/or added.

(Item 12A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus, andeverolimus.

(Item 13A)

The method of any one of items 1A to 12A, wherein the mTOR inhibitor isadministered as an eye drop.

(Item 14A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor israpamycin and is administered at a concentration of at least about 0.1nM.

(Item 15A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor isadministered as an eye drop, wherein the mTOR inhibitor is rapamycin andis present in the eye drop at at least about 0.1 mM.

(Item 16A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor istemsirolimus and is administered at a concentration of at least about0.01 nM.

(Item 17A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor isadministered as an eye drop, wherein the mTOR inhibitor is temsirolimusand is present in the eye drop at at least about 0.01 mM.

(Item 18A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor iseverolimus and is administered at a concentration of at least about 0.1nM.

(Item 19A)

The method of any one of items 1A to 8A, wherein the mTOR inhibitor isadministered as an eye drop, wherein the mTOR inhibitor is everolimusand is present in the eye drop at at least about 0.1 mM.

(Item 20A)

The method of any one of items 1A to 19A, wherein the mTOR inhibitor islocally administered.

(Item 21A)

The method of any one of items 1A to 20A, wherein the mTOR inhibitor islocally administered to an eye.

(Item 22A)

The method of any one of items 1A to 21A, wherein the mTOR inhibitor isadministered so as to contact a cornea.

(Item 1B)

Use of an mTOR inhibitor for manufacturing a medicament for preventingor treating an eye condition, disorder, or disease.

(Item 2B)

The use of item 1B, wherein the eye condition, disorder, or disease is acorneal endothelial condition, disorder, or disease.

(Item 3B)

The use of item 1B or 2B, wherein the eye condition, disorder, ordisease is a corneal endothelial condition, disorder, or disease due toa transforming growth factor-β (TGF-β).

(Item 4B)

The use of any one of items 1B to 3B, wherein the corneal endothelialcondition, disorder, or disease is selected from the group consisting ofFuchs' endothelial corneal dystrophy, post-corneal transplant disorder,corneal endotheliitis, trauma, ophthalmic surgery, post-ophthalmic lasersurgery disorder, aging, posterior polymorphous dystrophy (PPD),congenital hereditary endothelial dystrophy (CHED), idiopathic cornealendothelial disorder, and cytomegalovirus corneal endotheliitis.

(Item 5B)

The use of any one of items 1B to 4B, wherein the corneal endothelialcondition, disorder, or disease is due to overexpression ofextracellular matrix (ECM).

(Item 6B)

The use of item 5B, wherein the corneal endothelial condition, disorder,or disease is selected from the group consisting of Fuchs' endothelialcorneal dystrophy, guttae formation, hypertrophy of a Descemet'smembrane, hypertrophy of a cornea, turbidity, scar, corneal nebula,corneal macula, leucoma, photophobia, and blurred vision.

(Item 7B)

The use of any one of items 1B to 6B, wherein the condition, disorder,or disease comprises Fuchs' endothelial corneal dystrophy.

(Item 8B)

The use of any one of items 1B to 7B, wherein the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus,everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalisib,Ridaforolimus, NVP-BEZ235, CZ415, Torkinib, Torin 1, Omipalisib,OSI-027, PF-04691502, Apitolisib, WYE-354, Vistusertib, Torin 2,Tacrolimus, GSK1059615, Gedatolisib, WYE-125132, BGT226, Palomid 529,PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388,Zotarolimus, and Chrysophanic Acid.

(Item 9B)

The use of any one of items 1B to 7B, wherein the mTOR inhibitor is anmTOR gene expression suppressing substance.

(Item 10B)

The use of item 9B, wherein the mTOR gene expression suppressingsubstance is siRNA, antisense nucleic acid, or ribozyme against an mTORgene.

(Item 11B)

The use of item 9B or 10B, wherein the mTOR gene expression suppressingsubstance is siRNA against an mTOR gene, wherein the siRNA comprises asense strand consisting of a nucleic acid sequence set forth in SEQ IDNO: 1 or the nucleic acid sequence wherein 1 to 3 bases of nucleotidesare deleted, substituted, inserted and/or added, and an antisense strandconsisting of a nucleic acid sequence set forth in SEQ ID NO: 2 or thenucleic acid sequence wherein to 3 bases of nucleotides are deleted,substituted, inserted and/or added.

(Item 12B)

The use of any one of items 1B to 8B, wherein the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus, andeverolimus.

(Item 13B)

The use of any one of items 1B to 12B, wherein the medicament is an eyedrop.

(Item 14B)

The use of any one of items 1 to 8, wherein the mTOR inhibitor israpamycin and is present in the medicament at at least about 0.1 nM.

(Item 15B)

The use of any one of items 1B to 8B, wherein the medicament is an eyedrop, wherein the mTOR inhibitor is rapamycin and is present in the eyedrop at at least about 0.1 mM.

(Item 16B)

The use of any one of items 1B to 8B, wherein the mTOR inhibitor istemsirolimus and is present in the medicament at at least about 0.01 nM.

(Item 17B)

The use of any one of items 1B to 8B, wherein the medicament is an eyedrop, wherein the mTOR inhibitor is temsirolimus and is present in theeye drop at at least about 0.01 mM.

(Item 18B)

The use of any one of items 1B to 8B, wherein the mTOR inhibitor iseverolimus and is present in the medicament at at least about 0.1 nM.

(Item 19B)

The use of any one of items 1B to 8B, wherein the medicament is an eyedrop, wherein the mTOR inhibitor is everolimus and is present in the eyedrop at at least about 0.1 mM.

(Item 1C)

An mTOR inhibitor for use in preventing or treating an eye condition,disorder, or disease.

(Item 2C)

The mTOR inhibitor of item 1C, comprising one or more features of theitems above.

(Item 1D)

A composition for preserving corneal endothelial cells, comprising anmTOR inhibitor.

(Item 2D)

The composition of item 1D, comprising one or more features of the itemsabove.

(Item 1E)

A method for preserving corneal endothelial cells, comprising contactingan effective amount of an mTOR inhibitor with corneal endothelial cells.

(Item 2E)

The method of item 1E, comprising one or more features of the itemsabove.

(Item 1F)

A method for growing corneal endothelial cells or promoting growth ofcorneal endothelial cells, encompassing contacting an effective amountof an mTOR inhibitor with corneal endothelial cells.

(Item 2F)

The method of item 1E, comprising one or more features of the itemsabove.

The present invention is intended so that one or more of theaforementioned features can be provided not only as the explicitlydisclosed combinations, but also as other combinations thereof.Additional embodiments and advantages of the present invention arerecognized by those skilled in the art by reading and understanding thefollowing detailed description, as needed.

Advantageous Effects of Invention

The present invention unexpectedly discovered that an mTOR inhibitor maytreat or prevent a disease due to a disorder or a disease due totransforming growth factor-β (TGF-β) in Fuchs' endothelial cornealdystrophy or the like and can provide a medicament that may treat orprevent an eye condition, disorder, or disease including such a disease.The present invention also provides a medicament that can treat orprevent a disease, due to a corneal endothelial disorder due tooverproduction of extracellular matrix (e.g., fibronectin), such asguttae, hypertrophy of the Descemet's membrane, corneal turbidity,leucoma or other conditions of clouding. The present invention furtherprovides a composition for preserving corneal endothelial cells or acomposition for promoting the growth of corneal endothelial cells,comprising an mTOR inhibitor, as well as a method for preserving and/orgrowing or promoting the growth of corneal endothelial cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows microscopic images of iFECDs. The top left image shows acontrol group wherein TGF-β2 is not supplemented, the top right imageshows a group wherein TGF-β2 is supplemented, the bottom left imageshows a group wherein TGF-β2 and rapamycin are supplemented, and thebottom right image shows a group wherein TGF-β2 and Z-VD-FMK, which is acaspase inhibitor, are supplemented.

FIG. 2 shows the results of western blot on total caspase 3, cleavedcaspase 3, PARP and GAPDH. The figure shows, from the left, a controlgroup wherein TGF-β2 is not supplemented, a group wherein TGF-β2 issupplemented, a group wherein TGF-β2 and rapamycin are supplemented, anda group wherein TGF-β2 and Z-VD-FMK, which is a caspase inhibitor, aresupplemented.

FIG. 3 shows the results of western blot on Akt, p-Akt, S6K, p-S6K, andGAPDH. The figure shows, from the left, a control group wherein TGF-β2is not supplemented, a group wherein TGF-β2 is supplemented, a groupwherein TGF-β2 and rapamycin are supplemented, and a group whereinTGF-β2 and Z-VD-FMK, which is a caspase inhibitor, are supplemented.

FIG. 4 shows the results of western blot on Smad2, p-Smad2, Smad3,p-Smad3, and GAPDH. The figure shows, from the left, a control groupwherein TGF-β2 is not supplemented, a group wherein TGF-β2 issupplemented, a group wherein TGF-β2 and rapamycin are supplemented, anda group wherein TGF-β2 and Z-VD-FMK, which is a caspase inhibitor, aresupplemented.

FIG. 5 shows the results of western blot on fibronectin and GAPDH (n=3).The figure shows, from the left, a control group wherein TGF-β2 is notsupplemented, a group wherein TGF-β2 is supplemented, a group whereinTGF-β2 and rapamycin are supplemented, and a group wherein TGF-β2 andZ-VD-FMK, which is a caspase inhibitor, are supplemented.

FIG. 6 shows the results of agarose gel electrophoresis and westernblot. The top panel shows the result of the agarose gel electrophoresison mTOR and GAPDH and the bottom panel shows the results of the westernblot on mTOR, S6K, p-S6K, and GAPDH. The figure shows, from the left ofeach panel, a Random siRNA-supplemented group, a TGF-β2+RandomsiRNA-supplemented group, an mTOR siRNA-supplemented group, and aTGF-β2+mTOR siRNA-supplemented group.

FIG. 7 shows microscopic images of iFECDs. The top left image shows aRandom siRNA-supplemented group, the top right image shows an mTORsiRNA-supplemented group, the bottom left image shows a TGF-β2+RandomsiRNA-supplemented group, and the bottom right image shows a TGF-β2+mTORsiRNA-supplemented group.

FIG. 8 shows the results of western blot on total caspase 3, cleavedcaspase 3, PARP and GAPDH. The figure, from the left, shows a RandomsiRNA-supplemented group, a TGF-β2+Random siRNA-supplemented group, anmTOR siRNA-supplemented group, and a TGF-β2+mTOR siRNA-supplementedgroup.

FIG. 9 shows the results of western blot on fibronectin and GAPDH. Thefigure, from the left, shows a Random siRNA-supplemented group, aTGF-β2+Random siRNA-supplemented group, an mTOR siRNA-supplementedgroup, and a TGF-β2+mTOR siRNA-supplemented group.

FIG. 10 shows a graph of caspase 3/7 activity. The figure, from theleft, shows a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+rapamycin (0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1nM, 10 nM, 100 nM), and TGF-β2 (10 ng/ml)+Z-VD-FMK (10 μM). Thestatistical significance was tested by the Dunnett-t test (* indicatesp<0.05 and ** indicates p<0.01. n=5).

FIG. 11 shows a graph of caspase 3/7 activity. The figure shows, fromthe left, a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+everolimus (0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM), and TGF-β2 (10ng/ml)+Z-VD-FMK (10 μM). The statistical significance was tested by theDunnett-t test (* indicates p<0.05 and ** indicates p<0.01. n=5).

FIG. 12 shows a graph of caspase 3/7 activity. The figure, from theleft, shows a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+temsirolimus (0.000001 μM, 0.00001 μM, 0.0001 μM, 0.001 μM, 0.01μM, 0.1 μM, 1 μM, 10 μM), and TGF-β2+Z-VD-FMK (10 μM). The statisticalsignificance was tested by the Dunnett-t test (* indicates p<0.05 and **indicates p<0.01. n=5).

FIG. 13 shows a graph of caspase 3/7 activity. The figure, from theleft, shows a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+Pl-103 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β2 (10ng/ml)+Z-VD-FMK (10 μM). The statistical significance was tested by theDunnett-t test (* indicates p<0.05 and ** indicates p<0.01. n=5).

FIG. 14 shows a graph of caspase 3/7 activity. The figure shows, fromthe left, a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+CC-223 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β2 (10ng/ml)+Z-VD-FMK (10 μM). The statistical significance was tested by theDunnett-t test (* indicates p<0.05 and ** indicates p<0.01. n=5).

FIG. 15 shows a graph of caspase 3/7 activity. The figure shows, fromthe left, a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+INK128 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β2 (10ng/ml)+Z-VD-FMK (10 μM). The statistical significance was tested by theDunnett-t test (* indicates p<0.05 and ** indicates p<0.01. n=5).

FIG. 16 shows a graph of caspase 3/7 activity. The figure shows, fromthe left, a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+AZD8055 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β (10 ng/ml)2+Z-VD-FMK (10 μM). The statistical significance was tested by theDunnett-t test (* indicates p<0.05 and ** indicates p<0.01. n=5).

FIG. 17 shows a graph of caspase 3/7 activity. The figure shows, fromthe left, a TGF-β2-non-supplemented group, a control group, TGF-β2 (10ng/ml)+KU 0063794 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β2 (10ng/ml)+Z-VD-FMK (10 μM). The statistical significance was tested by theDunnett-t test (* indicates p<0.05 and ** indicates p<0.01. n=5).

FIG. 18 shows a schematic diagram of the relationship of vision withcorneal endothelial cells and ECM deposition. The diagram shows thatvision deteriorates as corneal endothelial cells and ECM depositionincreases. Hypertrophy of the Descemet's membrane or guttae due to ECMdeposition commonly starts developing in the age of 30s and 40s inFuchs' endothelial corneal dystrophy patients, and progresses throughoutthe patients' lives. Progression results in visual impairment such asblurred vision, halo, glare, or reduced vision. While cornealendothelial cell death progresses concurrently, the transparency of acorneal is maintained by the remaining corneal endothelia compensatingfor the pumping function until the corneal endothelial cell density isbelow about 1000 cells/mm². If the density is below about 1000cells/mm², infiltration of the anterior aqueous humor into the cornealeads to corneal edema, resulting in severe visual impairment. Thepresent technique can maintain visual function by suppressing both ECMdeposition and corneal endothelial cell death.

FIG. 19 shows a typical example of a corneal endothelial cell imageobserved by a contact corneal endothelial specular microscopy using anFECD model mouse, in which 2 μl of mTOR inhibitor eye drop (1 mM) wasinstilled into each of the left and right eyes twice a day in themorning and in the evening for two months. An image whereinphysiological saline (base) was instilled is shown as a control.

FIG. 20 shows a graph of the corneal endothelial cell density (cell/mm²)in an FECD model mouse, in which an mTOR inhibitor eye drop wasinstilled. A graph wherein physiological saline (base) was instilled isshown as a control. The statistical significance was tested by theStudent's t test (* indicates p<0.05. n=3).

FIG. 21 shows a graph of the area (%) of guttae in an FECD model mouse,in which an mTOR inhibitor eye drop was instilled. A graph whereinphysiological saline (base) was instilled is shown as a control. Thestatistical significance was tested by the Student's t test (**indicates p<0.01. n=3).

DESCRIPTION OF EMBODIMENTS

The present invention is explained hereinafter. Throughout the entirespecification, a singular expression should be understood asencompassing the concept thereof in the plural form, unless specificallynoted otherwise. Thus, singular articles (e.g., “a”, “an”, “the”, andthe like in the case of English) should also be understood asencompassing the concept thereof in the plural form, unless specificallynoted otherwise. Further, the terms used herein should be understood asbeing used in the meaning that is commonly used in the art, unlessspecifically noted otherwise. Therefore, unless defined otherwise, allterminologies and scientific technical terms that are used herein havethe same meaning as the general understanding of those skilled in theart to which the present invention pertains. In case of a contradiction,the present specification (including the definitions) takes precedence.

Definition

As used herein, “about” before a numerical value means±10% of thenumerical value that follows.

As used herein, “mTOR inhibitor” refers to any agent that inhibitssignaling of mTOR. An mTOR inhibitor is preferably water-soluble. Thisis because, unless an mTOR inhibitor is water-soluble, it may benecessary to use a solvent that is not highly biocompatible.Water-solubility can be classified based on the definition of solubilityin the pharmacopoeia. In other words, the amount of solvent required todissolve 1 g or 1 mL of solute is defined as extremely readilydissolvable: less than 1 mL; readily dissolvable: 1 mL or greater andless than 10 mL; somewhat readily dissolvable: 10 mL or greater and lessthan 30 mL; somewhat difficult to dissolve: 30 mL or greater and lessthan 100 mL; difficult to dissolve: 100 mL or greater and less than 1000mL; very difficult to dissolve: 1000 mL or greater and less than 10000mL; and hardly dissolvable: 10000 mL or greater. Solubility is similarlyassessed herein. Water solubility is understood to mean that a substancewith any solubility can be used, as long as an effective amount thereofcan be dissolved when water is used as a solvent. Such a water-solublecomponent is advantageously used as an eye drop.

An mTOR (mammalian target of rapamycin) is a serine/threonine kinaseidentified as a target molecule of rapamycin and is considered to play acentral role in the adjustment of cell division, survival and the like.An mTOR is also known as SKS; FRAP; FRAP1; FRAP2; RAFT1; RAPT1, and 2475is given as a Gene ID of NCBI. Based on such information, those skilledin the art can design and manufacture various mTOR inhibitors.

The mTOR inhibitors that can be used in the present invention are notparticularly limited, as long as they are compounds having mTORinhibiting activity. Examples thereof include, rapamycin, temsirolimus,everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalisib(XL765, SAR245409), Ridaforolimus (Deforolimus, MK-8669), NVP-BEZ235,CZ415, Torkinib (PP242), Torin 1, Omipalisib (GSK2126458, GSK458),OSI-027, PF-04691502, Apitolisib (GDC-0980, RG7422), WYE-354,Vistusertib (AZD2014), Torin 2, Tacrolimus (FK506), GSK1059615,Gedatolisib (PF-05212384, PKI-587), WYE-125132 (WYE-132), BGT226(NVP-BGT226), Palomid 529 (P529), PP121, WYE-687, CH5132799, WAY-600,ETP-46464, GDC-0349, XL388, Zotarolimus (ABT-578), and ChrysophanicAcid.

Preferred mTOR inhibitors include, but are not limited to, rapamycin,temsirolimus and everolimus. Although not wishing to be bound by anytheory, this is because these pharmaceutical products are approved byFDA, PMDA, and the like and problems in the aspects of safety andtoxicity are minimized. An ever more preferable mTOR inhibitor israpamycin. Another preferable mTOR inhibitor is temsirolimus. Anotherpreferable mTOR inhibitor is, but is not limited to, everolimus.

Other examples of mTOR inhibitors that can be used in the presentinvention include neutralizing antibodies against mTORs, compoundsinhibiting the activity of mTORs, compounds inhibiting the transcriptionof a gene encoding an mTOR (e.g., antisense nucleic acids, siRNAs,ribozymes), peptides, compounds with a plant component, a component oftraditional medicament such as Kampo medicine, or other components, andthe like.

Antisense nucleic acids used in the present invention may inhibit theexpression and/or function of a gene (nucleic acids) encoding a memberof a signaling pathway of an mTOR or the like by any of theabove-described action. As one embodiment, designing an antisensesequence complementary to an untranslated region near the 5′ end of mRNAof a gene encoding the aforementioned mTOR is considered effective forinhibiting translation of a gene. Further, a sequence that iscomplementary to an untranslated region of 3′ or a coding region canalso be used. In this manner, antisense nucleic acids utilized in thepresent invention include not only a translation region of a geneencoding the aforementioned mTOR or the like, but also nucleic acidscomprising an antisense sequence of a sequence of an untranslatedregion. An antisense nucleic acid to be used is linked to the downstreamof a suitable promoter, and preferably a sequence comprising atranscription termination signal is linked to the 3′ side. A nucleicacid prepared in this manner can be transformed into a desired animal(cell) by using a known method. A sequence of an antisense nucleic acidis preferably a sequence that is complementary to a gene encoding anmTOR of the animal (cell) to be transformed or a portion thereof.However, such a sequence does not need to be fully complementary, aslong as gene expression can be effectively suppressed. A transcribed RNApreferably has complementarity that is 90% or greater, and mostpreferably 95% or greater, with respect to a transcript of a targetgene. In order to effectively inhibit the expression of a target geneusing an antisense nucleic acid, it is preferable that the length of theantisense nucleic acid is at least 12 bases and less than 25 bases.However, the antisense nucleic acid of the present invention is notnecessarily limited to this length. For example, the length may be 11bases or less, 100 bases or more, or 500 bases or more. An antisensenucleic acid may be composed of only DNA, but may comprise a nucleicacid other than DNAs, such as a locked nucleic acid (LNA). As oneembodiment, an antisense nucleic acid used in the present invention maybe an LNA containing antisense nucleic acid comprising LNA at the 5′ endor LNA at the 3′ end. In an embodiment using an antisense nucleic acidin the present invention, the antisense sequence can be designed basedon a nucleic acid sequence of an mTOR by using the method described in,for example, Hirashima and Inoue, Shin-seikagaku Jikkenn Kouza 2 [NewBiochemical Experiment Course 2] Kakusan IV Idenshi no Fukusei toHatsugen [Duplication and Expression of Gene of Nucleic Acid IV], Ed. bythe Japanese Biochemical Society, Tokyo Kagaku Dojin, 1993, 319-347.

Expression of mTOR can also be inhibited by utilizing a ribozyme or DNAencoding a ribozyme. A ribozyme refers to an RNA molecule havingcatalytic activity. While there are ribozymes with various activities, astudy focusing on especially ribozymes as an enzyme for cleaving an RNAmade it possible to design a ribozyme that site-specifically cleaves anRNA. There are ribozymes with a size of 400 nucleotides or more as ingroup I intron ribozymes and M1 RNA contained in RNase P, but there arealso those with an active domain of about 40 nucleotides calledhammerhead or hair-pin ribozymes (Makoto Koizumi and Eiko Otsuka,Protein, Nucleic Acid and Enzyme, 1990, 35, 2191).

For example, a self-cleaving domain of a hammerhead ribozyme cleaves the3′ side of C15 of a sequence called G13U14C15. Base pair formation ofU14 and A9 is considered important for the activity thereof. It is alsodemonstrated that cleavage can also be made at A15 or U15 instead of C15(Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) Restriction enzyme-likeRNA-cleaving ribozymes that recognize the sequence UC, UU, or UA in thetarget RNAs can be created by designing their substrate-binding sites tobe complementary to an RNA sequence near the target site (Koizumi, M. etal., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka,Protein, Nucleic Acid and Enzyme, 1990, 35, 2191., Koizumi, M. et al.,Nucl. Acids Res., 1989, 17, 7059.)

Hairpin ribozymes are also useful for the objective of the presentinvention. Such a ribozyme is found, for example, in the minus strand ofa tobacco ringspot virus satellite RNA (Buzayan, J M., Nature, 1986,323, 349). It is demonstrated that target specific RNA-cleavingribozymes can also be created from hairpin ribozymes (Kikuchi, Y. &Sasaki, N., Nucl. Acids Res, 1991, 19, 6751., Yo Kikuchi, Kagaku toSeibutsu [Chemistry and Biology], 1992, 30, 112). In this manner,expression of a gene encoding an mTOR or the like can be inhibited byspecifically cleaving a transcript of the gene by using a ribozyme.

Expression of an endogenous gene of an mTOR can also be suppressed byRNA interference (hereinafter, abbreviated as “RNAi”) using adouble-stranded RNA having a sequence that is identical or similar to atarget gene sequence. RNAi is a methodology that is currently drawingattention, which can suppress the expression of a gene having a sequencethat is homologous to a double-stranded RNA (dsRNA) when the dsRNA isincorporated directly into a cell. In mammalian cells, short strandeddsRNA (siRNA) can be used to induce RNAi. RNAi has many advantages overknockout mice, such as a stable effect, facilitated experiment, and lowcost. SiRNA is discussed in detail in other parts of the specification.

As used herein “siRNA” is an RNA molecule having a double-stranded RNAportion consisting of 15 to 40 bases, where siRNA has a function ofcleaving mRNA of a target gene with a sequence complementary to anantisense strand of the siRNA to suppress the expression of the targetgene. Specifically, the siRNA in the present invention is an RNAcomprising a double-stranded RNA portion consisting of a sense RNAstrand consisting of a sequence homologous to consecutive RNA sequencesin mRNA of mTOR and an antisense RNA strand consisting of a sequencecomplementary to the sense RNA sequence. Design and manufacture of suchsiRNA and mutant siRNA discussed below are within the technicalcompetence of those skilled in the art. Any consecutive RNA regions ofmRNA which is a transcript of a sequence of mTOR can be appropriatelyselected to make double-stranded RNA corresponding to this region, whichis within the ordinary procedure performed by those skilled in the art.Further, those skilled in the art can appropriately select an siRNAsequence having a stronger RNAi effect from mRNA sequences, which aretranscripts of the sequence, by a known method. Further, if one of thestrands is revealed, those skilled in the art can readily find the basesequence of the other stand (complementary strand). SiRNA can beappropriately made by using a commercially available nucleic acidsynthesizer. A common synthesis service can also be utilized for desiredRNA synthesis.

In terms of bases, the length of a double-stranded RNA portion is 15 to40 bases, preferably 15 to 30 bases, more preferably 15 to 25 bases,still more preferably 18 to 23 bases, and most preferably 19 to 21bases. It is understood that the upper limits and the lower limitsthereof are not limited to such specific limits, and may be of anycombination of the mentioned limits. The end structure of a sense strandor antisense strand of siRNA is not particularly limited, and can beappropriately selected in accordance with the objective. For example,such an end structure may have a blunt end or a sticky end (overhang). Atype where the 3′ end protrudes out is preferred. SiRNA having anoverhang consisting of several bases, preferably 1 to 3 bases, and morepreferably 2 bases at the 3′ end of a sense RNA strand and antisense RNAstrand is preferable for having a large effect of suppressing expressionof a target gene in many cases. The type of bases of an overhang is notparticularly limited, which may be either a base constituting an RNA ora base constituting a DNA. An example of a preferred overhang sequenceincludes dTdT at the 3′ end (2 bp of deoxy T) and the like. Examples ofpreferable siRNA include, but are not limited to, all siRNAs with dTdT(2 bp of deoxy T) at the 3′ end of the sense or antisense strands of thesiRNA.

Furthermore, it is also possible to use siRNA in which one to severalnucleotides are deleted, substituted, inserted and/or added at one orboth of the sense strand and antisense strand of the siRNA describedabove. One to several bases as used herein is not particularly limited,but preferably refers to 1 to 4 bases, more preferably 1 to bases, andmost preferably 1 to 2 bases. Specific examples of such mutationsinclude, but are not limited to, mutations resulting in 0 to 3 bases atthe 3′-overhang portion, mutations that change the base sequence of the3′-overhang portion to another base sequence, mutations in which thelengths of the above-described sense RNA strand and antisense RNA strandare different by 1 to 3 bases due to insertion, addition or deletion ofbases, mutations substituting a base in the sense strand and/or theantisense with another base, and the like. However, it is necessary thatthe sense strand and the antisense strand can hybridize in such mutantsiRNAs, and these mutant siRNAs have the ability to suppress geneexpression that is equivalent to that of siRNAs without any mutations.

SiRNA may also be a molecule with a structure in which one end isclosed, such as siRNA with a hairpin structure (Short Hairpin RNA;shRNA). A shRNA is an RNA comprising a sense strand RNA with a specificsequence of a target gene, an antisense strand RNA consisting of asequence complementary to the sense strand sequence, and a linkersequence for connecting the two strands, wherein the sense strandportion hybridizes with the antisense strand portion to form adouble-stranded RNA portion.

It is desirable for siRNA to not exhibit the so-called off-target effectin clinical use. An off-target effect refers to an action forsuppressing the expression of another gene, besides the target gene,which is partially homologous to the siRNA used. In order to avoid anoff-target effect, it is possible to confirm that a candidate siRNA doesnot have cross reactivity by using a DNA microarray or the like inadvance. Further, it is possible to avoid an off-target effect byconfirming whether there is a gene comprising a moiety that is highlyhomologous to a sequence of a candidate siRNA, other than a target gene,using a known database provided by the NCBI (National Center forBiotechnology Information) or the like.

In order to make the siRNA according to the present invention, a knownmethod, such as a method using chemical synthesis or a method using agene recombination technique, can be appropriately used. With a methodusing synthesis, a double-stranded RNA can be synthesized based onsequence information by using a common method. With a method using agene recombination technique, a siRNA can be made by constructing anexpression vector encoding a sense strand sequence or an antisensestrand sequence and introducing the vector into a host cell, and thenobtaining each of sense strand RNA and antisense strand RNA produced bytranscription. It is also possible to make a desired double-stranded RNAby expressing an shRNA forming a hairpin structure, comprising a sensestrand of a specific sequence of a target gene, an antisense strandconsisting of a sequence complementary to the sense strand sequence, anda linker sequence for linking the two strands.

For a siRNA, all or part of the nucleic acid constituting the siRNA maybe a natural or a modified nucleic acid, as long as such a nucleic acidhas activity to suppress the expression of a target gene.

The siRNA according to the present invention does not necessarily haveto be a pair of double-stranded RNAs to a target sequence. It may be amixture of a plurality of pairs (the “plurality” is not particularlylimited, but preferably refers to a small number of about 2 to 5) ofdouble-stranded RNAs to a region comprising a target sequence. In thisregard, those skilled in the art can appropriately make an siRNA as anucleic acid mixture corresponding to a target sequence by using acommercially available nucleic acid synthesizer and a DICER enzyme. Acommon synthesis service can also be utilized for desired RNA synthesis.It should be noted that the siRNA according to the present inventionencompasses the so-called “cocktail siRNA”. For the siRNA according tothe present invention, not all the nucleotides have to be aribonucleotide (RNA). In other words, in the present invention, one orplurality of ribonucleotides constituting an siRNA may be acorresponding deoxyribonucleotide. This term “corresponding” refers tohaving the same base type (adenine, guanine, cytosine, thymine (uracil))but a different sugar moiety structure. For example, adeoxyribonucleotide corresponding to a ribonucleotide having adeninerefers to a deoxyribonucleotide having adenine.

Furthermore, a DNA (vector) which can express the above-described RNAaccording to the present invention is also encompassed as a preferredembodiment of a nucleic acid which can suppress the expression of anmTOR or the like. For example, the DNA (vector) which can express theabove-described double-stranded RNA according to the present inventionis a DNA having a structure in which a DNA encoding one of the strandsof the double-stranded RNA and a DNA encoding the other strand of thedouble-stranded RNA are linked to a promoter so that each of the DNAscan be expressed. The above-described DNA according to the presentinvention can be appropriately made by those skilled in the art by usinga common genetic engineering technique. More specifically, theexpression vector according to the present invention can be made byappropriately inserting a DNA encoding the RNA of interest into variousknown expression vectors.

In the present invention, a modified nucleic acid may be used as anucleic acid for suppressing the expression of a target gene. A modifiednucleic acid refers to a nucleic acid, which has a modification at anucleoside (base moiety, sugar moiety) and/or an inter-nucleosidebinding site and has a structure that is different from that of anaturally occurring nucleic acid. Examples of “modified nucleoside”constituting a modified nucleic acid include: abasic nucleosides;arabinonucleoside, 2′-deoxyuridine, α-deoxyribonucleoside,β-L-deoxyribonucleoside, and other sugar modification bearingnucleosides; peptide nucleic acids (PNA), phosphate group-bindingpeptide nucleic acids (PHONA), locked nucleic acids (LNA), morpholinonucleic acids and the like. The above sugar modification bearingnucleosides include 2′-O-methylribose, 2′-deoxy-2′-fluororibose,3′-O-methylribose and other substituted pentose; 1′,2′-deoxyribose;arabinose; substituted arabinose sugar; and nucleoside having a sugarmodification of alpha-anomer and hexose. These nucleosides may be amodified base in which the base moiety is modified. Examples of suchmodified bases include pyrimidine such as 5-hydroxycytosine,5-fluorouracil, and 4-thiouracil; purine such as 6-methyladenine and6-thioguanosine; other heterocyclic bases and the like.

Examples of a “modified inter-nucleoside bond” which constitutes amodified nucleic acid include alkyl linker, glyceryl linker, aminolinker, poly(ethylene glycol) bond, inter-methyl phosphonate nucleosidebond; and bonds between non-natural nucleosides such asmethylphosphonothioate, phosphotriester, phosphothiotriester,phosphorothioate, phosphorodithioate, triester prodrug, sulfone,sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate,carbamate, morpholino, boranophosphonate, and phosphoramidate.

The nucleic acid sequence comprised in the double-stranded siRNAaccording to the present invention includes siRNAs for an mTOR, othermTOR signaling members and the like.

It is also possible to introduce the nucleic acid or agent according tothe present invention into a phospholipid endoplasmic reticulum such asa liposome and administer the endoplasmic reticulum. An endoplasmicreticulum in which an siRNA or shRNA is retained can be introduced intoa predetermined cell using lipofection. The resulting cell is thensystemically administered, such as intravenously, intra-arterially orthe like. It can also be locally administered to a required site in aneye or the like. While an siRNA exhibits a very good specific,post-transcription suppressing effect in vitro, the siRNA is quicklydegraded in vivo due to nuclease activity in the serum so that theduration thereof is limited. Therefore, there has been a need for thedevelopment of a better and more effective delivery system. As anexample, Ochiya, T et al., Nature Med., 5: 707-710, 1999, Curr. GeneTher., 1: 31-52, 2001 reports that a biocompatible materialatelocollagen, when mixed with a nucleic acid to form a complex, is acarrier which has an action of protecting a nucleic acid from adegrading enzyme in a living organism and is extremely suitable as acarrier for an siRNA. While such a form can be used, the method forintroducing a nucleic acid, therapeutic or prophylactic drug accordingto the present invention is not limited thereto. In this manner, due tothe fast degradation by the action of a nuclease in the serum in aliving organism, it becomes possible to achieve continuation of aneffect for an extended period of time. For example, Takeshita F. PNAS,(2003) 102 (34) 12177-82, Minakuchi Y Nucleic Acids Research (2004) 32(13) e109 report that atelocollagen derived from bovine skin forms acomplex with a nucleic acid, which has action of protecting a nucleicacid from a degrading enzyme in a living organism and is extremelysuitable as a carrier for an siRNA. Such a technique can be used.

As used herein, “iFECD” (immortalized Fuchs' endothelial cornealdystrophy) is an abbreviation for immortalized Fuchs' endothelialcorneal dystrophy cells. A manufacturing method of iFECD is describedin, for example, WO 2015/015655.

As used herein, “HCEC” (human corneal endothelial cells) is anabbreviation for human corneal endothelial cells. In addition, “iHCEC”is an abbreviation for immortalized human corneal endothelial cells.

As used herein, “programmed cell death” refers to a phenomenon of cellsspontaneously dying at a determined time or environment as if the deathis pre-programmed. Programmed cell death is used in the meaning thatincludes, for example, “apoptosis”.

As used herein, “transforming growth factor-β (also denoted with theabbreviation TGF-β)” is used in the same meaning as those used in theart. It is a homodimer multifunctional cytokine with a molecular weightof 25 kD exhibiting a variety of biological activity, such as beingresponsible for pathogenesis of various sclerotic diseases, rheumatoidarthritis, and proliferative vitreoretinopathy, being deeply involved inhair loss, suppressing the functioning of immunocompetent cells whilesuppressing overproduction of protease to prevent degradation ofpulmonary tissue resulting in pulmonary emphysema, and suppressingcancer cell growth. “TGF-β signal” refers to a signal mediated by TGF-β,which is elicited by TGF-β. Examples of TGF-β signals include signalsmediated by TGF-β2 in addition to signals mediated by TGF-β1, TGF-β3 orthe like. In humans, TGF-β has three isoforms, TGF-β1 to β3, which havehomology of about 70% and similar action. TGF-β is produced as aninactive latent form with a molecular weight of about 300 kD which isunable to bind to a receptor. The action thereof is exerted by beingactivated on a target cell surface or the surroundings thereof to becomean active form that can bind to a receptor.

Although not wishing to be bound by any theory, the action of TGF-β in atarget cell is understood to be transmitted by a phosphorylation pathwayof a series of proteins responsible for transmitting information calledSmad. First, when activated TGF-β binds to a TGF-β type II receptor on atarget cell surface, a receptor complex consisting of two molecules oftype II receptors and two molecules of TGF-β type I receptors is formed,and the type II receptors phosphorylate the type I receptors. It isunderstood that when the phosphorylated type I receptors phosphorylateSmad2 or Smad3, the phosphorylated Smad2 or Smad3 forms a complex withSmad4, where the complex migrates to a nucleus and binds to a targetsequence called CAGA box that is present in a target gene promotorregion to induce transcription and expression of a target gene with acoactivator.

A transforming growth factor-β (TGF-β) signaling pathway can modulatemany cellular activities, such as cell growth and differentiation,growth arrest, programmed cell death, and epithelial mesenchymaltransition (EMT), by modulating the target gene. Members of the TGF-βfamily including TGF-β itself (e.g., TGF-β1, TGF-β2, and TGF-β3),activin, and bone morphogenetic proteins (BMP) are potent modulators ofcell growth, differentiation, migration, programmed cell death, and thelike.

TGF-β is a protein of about 24 Kd produced by many cells including Blymphocytes, T lymphocytes, and activated macrophages and by many othercell types. Effects of TGF-β on the immune system include IL-2 receptorinduction, inhibition of IL-1 induced thymocyte growth, and blocking ofIFN-γ induced macrophage activation. TGF-β is considered to be involvedin various pathological conditions (Border et al. (1992) J. Clin.Invest. 90:1) and is thoroughly proven to function as either a tumorsuppressing substance or a tumor promotor.

Signaling of TGF-β is mediated by two serine/threonine kinase cellsurface receptors TGF-βRII and ALK5. TGF-β signaling is initiated byligand induced receptor dimerization enabling TGF-βRII to phosphorylatean ALK5 receptor. The phosphorylation activates ALK5 kinase activity,and the activated ALK5 then phosphorylates a downstream effector Smadprotein (vertebrate homologue of MAD or “Mothers against DPP(decapentaplegic)” protein), Smad2 or Smad3. A p-Smad2/3 complex withSmad4 enters a nucleus and activates transcription of a target gene.

Smad3 is a member of the R-Smad (receptor-activated Smad) subgroup ofSmad and a direct mediator of transcription activation by a TGF-βreceptor. A TGF-β stimulation results in phosphorylation and activationof Smad2 and Smad3, which form a complex with Smad4 (“common Smad” or“co-Smad” in vertebrates). This accumulates with the nucleus andmodulates transcription of a target gene. R-Smad is localized in acytoplasm and forms a complex with co-Smad through ligand inducedphosphorylation by a TGF-β receptor, migrates to the nucleus, where itmodulates gene expression associated with a cooperative transcriptionfactor and chromatin. Smad6 and Smad7 are inhibitory Smad (“I-Smad”),i.e., they are transcriptionally induced by TGF-β and function as aTGF-β signaling inhibitor (Feng et al. (2005) Annu. Rev. Cell. Dev.Biol. 21: 659). Smad6/7 obstruct receptor-mediated activation of R-Smadto exert an inhibitory effect thereof; and they are associated with atype I receptor, which competitively obstructs mobilization andphosphorylation of R-Smad. Smad6 and Smad7 are known to replenish E3ubiquitin ligase, which induces ubiquitination and degradation ofSmad6/7 interacting proteins.

TGF-β signaling pathways also have other pathways using transmission byBMP-7 or the like, which go through ALK-1/2/3/6 to express a functionvia Smad1/5/8. For TGF-β signaling pathways, see J. Massagu'e, Annu.Rev. Biochem. 1998. 67: 753-91; Vilar J M G, Jansen R, Sander C (2006)PLoS Comput Biol 2(1): e3; Leask, A., Abraham, D. J. FASEB J. 18,816-827 (2004); Coert Margadant & Arnoud Sonnenberg EMBO reports (2010)11, 97-105; Joel Rosenbloom et al., Ann Intern Med. 2010; 152: 159-166and the like.

As used herein, “eye condition, disorder, or disease” refers to anycondition, disorder, or disease in an eye. An eye condition, disorder,or disease is a condition, disorder, or disease in, for example, theretina, vitreous humor, lens, cornea, sclera, or different portion ofthe eye. The mTOR inhibitor in the present invention may be especiallyeffective against corneal endothelial condition, disorder, or disease.

As used herein, “corneal endothelial condition, disorder, or disease dueto transforming growth factor-β (TGF-β)” refers to any cornealendothelial condition, disorder, or disease induced by TGF-β in cornealendothelial cells. In the present invention, exposure of cornealendothelial cells such as model cells of Fuchs' endothelial cornealdystrophy (e.g., iFECD) to TGF-β2 surprisingly resulted in variousdisorders (e.g., programmed cell death). This is a phenomenon that hadnot been well understood conventionally. The inventors, after furtheranalysis of the corneal endothelial condition, disorder, or disease dueto a TGF-β signal, unexpectedly discovered that this disorder can besuppressed with an mTOR inhibitor. A corneal endothelial condition,disorder, or disease due to a TGF-β signal is associated with adifferent signaling pathway of mTOR. Examples of corneal endothelialconditions, disorders, or diseases due to a TGF-β signal include, butare not limited to, Fuchs' endothelial corneal dystrophy, post-cornealtransplant disorder, corneal endotheliitis, trauma, post-ophthalmicsurgery disorder, post-ophthalmic laser surgery disorder, aging,posterior polymorphous dystrophy (PPD), congenital hereditaryendothelial dystrophy (CHED), idiopathic corneal endothelial disorder,cytomegalovirus corneal endotheliitis and the like in which TGF-βexpression is observed. Since the disorder discovered in the presentinvention or a disorder associated therewith is considered expressed orraised especially in corneal endothelial cells or corneal endothelialtissue with higher than normal TGF-β2 expression, any cornealendothelial condition, disorder, or disease in which such cornealendothelial cells or corneal endothelial tissue are observed areespecially intended as the target of the present invention.

As used herein, “overexpression of extracellular matrix in cornealendothelial cells” refers to expression of extracellular matrix at anabnormal level compared to extracellular matrix expression levels innormal corneal endothelial cells. “Expression of extracellular matrix atan abnormal level” refers to production of extracellular matrix proteinssuch as fibronectin at an amount greater than the amount produced inextracellular matrix in a normal form. The production status includes nostimulation, as well as increased amount of expression due to a responseto transforming growth factor (TGF) β as needed. For example, this canbe about 1.1 fold or greater, about 1.2 fold or greater, about 1.3 foldor greater, about 1.4 fold or greater, about 1.5 fold or greater, about1.6 fold or greater, about 1.7 fold or greater, about 1.8 fold orgreater, about 1.9 fold or greater, or about 2.0 fold or greater withrespect to the amount of extracellular matrix under normal circumstancesfor human corneal endothelial cells. The difference relative to normalis preferably, but not necessarily, statistically significant. It issufficient that the difference is a medically significant difference.

As used herein, “corneal endothelial disorder due to overexpression ofextracellular matrix (ECM)” or a “condition” thereof is mainly adisorder associated with hypertrophy, deposition, clouding due toextracellular matrix or the like, or a condition thereof, which resultsin guttata on the corneal endothelium surface, hypertrophy of theDescemet's membrane such as turbid guttae of the Descemet's membrane, orthe like, and is associated with a condition that causes reduced vision.In corneal endothelial disorders such as Fuchs' corneal dystrophy,overproduction of extracellular matrix worsens the vision or visualsense even without a reduction in cell count, unlike exacerbation in acondition due to death (especially apoptosis) of corneal endothelialcells. Thus, even if cell death can be suppressed, this needs to beaddressed. “Corneal endothelial disorder due to overproduction ofextracellular matrix (ECM)” and “condition” thereof include, but are notlimited to, turbidity, scar, corneal nebula, corneal macula, cornealleucoma, and the like.

In a preferred embodiment, the conditions, disorders, or diseasestargeted by the present invention are disorders related to Fuchs'endothelial corneal dystrophy. It is demonstrated that TGF-β inductionin corneal endothelial cells is involved in Fuchs' endothelial cornealdystrophy. It is also demonstrated that TGF-β induction may be involvedin cell loss in FECDs. Therefore, inhibition of a TGF-β signalingpathway is naturally expected to be an effective therapy for FECDs.However, the inventors unexpectedly found that an mTOR inhibitor cansuppress a disorder due to a TGF-β signal.

Since the medicament of the present invention can treat cell damage orthe like that is induced by TGF-β2, which can be one of the major causesof abnormalities or disorders in Fuchs' endothelial corneal dystrophy,the medicament is understood to be useful in treating or preventingFuchs' endothelial corneal dystrophy. In particular, the presentinvention was able to suppress cell damage or programmed cell deathinduced by TGF-β2 in a Fuchs' endothelial corneal dystrophy model in theExamples, so that the present invention can be considered usable intherapy for patients with a severe TGF-β2 associated disease in a Fuchs'endothelial corneal dystrophy model. The medicament of the presentinvention can also, unexpectedly, suppress overexpression ofextracellular matrix (ECM), so that the medicament can treat or preventa disorder or the like in corneal endothelia such as ECM deposition inthe Descemet's membrane. Therefore, the present invention can treat orprevent damage to corneal endothelial cells in Fuchs' endothelialcorneal dystrophy, decreased corneal endothelial density, guttaeformation, hypertrophy of the Descemet's membrane, hypertrophy of acornea, corneal epithelial disorder, turbidity, scar, turbidity incorneal stroma, photophobia, blurred vision, visual impairment,ophthalmalgia, epiphora, hyperemia, pain, bullous keratopathy, eyediscomfort, diminished contrast, halo, glare, edema of the cornealstroma, and the like.

(General Techniques)

Molecular biological methodology, biochemical methodology,microbiological methodology used herein are well known andconventionally used in the art, which are described for example inSambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, ColdSpring Harbor and 3rd Ed. thereof (2001); Ausubel, F. M. (1987). CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience; Ausubel, F. M. (1989). Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A.(1990). PCR Protocols: A Guide to Methods and Applications, AcademicPress; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995).PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Wiley, and annual updates; Sninsky, J. J. et al.(1999). PCR Applications: Protocols for Functional Genomics, AcademicPress, Gait, M. J. (1985). Oligonucleotide Synthesis: A PracticalApproach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides andAnalogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992).The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. etal. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim;Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology,Oxford University Press; Hermanson, G. T. (1996). BioconjugateTechniques, Academic Press, Bessatsu Jikken Igaku [ExperimentalMedicine, Supplemental Volume], Idenshi Donyu & Hatsugen Kaiseki JikkenHo [Experimental Methods for Transgenesis & Expression Analysis],Yodosha, 1997, or the like. The reports by Nancy Joyce et al {Joyce,2004 #161} and {Joyce, 2003 #7} are well known for corneal endothelialcells. However, as discussed above, long-term culture or subcultureresults in fibroblast-like transformation, and research for an effectiveculturing method are currently ongoing. Relevant portions thereof (whichmay be the entire document) are incorporated herein by reference.

DESCRIPTION OF PREFERRED EMBODIMENTS

The preferred embodiments of the present invention are describedhereinafter. It is understood that the embodiments are exemplificationof the present invention, so that the scope of the present invention isnot limited to such preferred embodiments. It should be understood thatthose skilled in the art can refer to the following preferredembodiments to readily make modifications or changes within the scope ofthe present invention. Any of these embodiments of the present inventioncan be appropriately combined by those skilled in the art.

<Medicament>

In one aspect, the present invention provides a composition for use inpreventing or treating an eye condition, disorder, or disease,comprising an mTOR inhibitor. In particular, the mTOR inhibitor iseffective against a condition, disorder, or disease in cornealendothelia.

Although an mTOR is understood to be involved in signaling inside a celland is responsible for adjustment of cell mitosis, survival of a celland the like, the mechanism thereof in corneal endothelia is notelucidated. Thus, it was unexpected and surprising that an mTORinhibitor is effective in treating and preventing an ophthalmic,especially corneal endothelial, diseases, disorders, or conditions.

In one embodiment, a corneal endothelial condition, disorder, or diseasedue to transforming growth factor-β (TGF-β) in corneal endothelial cellsis selected from the group consisting of Fuchs' endothelial cornealdystrophy, post-corneal transplant disorder, corneal endotheliitis,trauma, post-ophthalmic surgery disorder, post-ophthalmic laser surgerydisorder, aging, posterior polymorphous dystrophy (PPD), congenitalhereditary endothelial dystrophy (CHED), idiopathic corneal endothelialdisorder, and cytomegalovirus corneal endotheliitis.

In still another preferred embodiment, the present invention provides amedicament having an action and effect for treating or preventing acondition due to overexpression of extracellular matrix in Fuchs'endothelial corneal dystrophy for use in treating or preventing such acondition, or a method of treating or preventing such a condition.Examples of such a condition include guttata on a corneal endothelialsurface, turbid guttae of a Descemet's membrane, hypertrophy of theDescemet's membrane, blurred vision, halo, glare, reduced vision,corneal turbidity, leucoma, abnormality in visual sense, and the like.Conditions due to overproduction of extracellular matrix are furtherdiscussed below.

In still another aspect, the present invention provides a medicament foruse in treating or preventing a corneal endothelial condition, disorder,or disease due to overexpression of extracellular matrix in cornealendothelial cells, comprising an mTOR inhibitor. As discussed above, anmTOR inhibitor can treat or prevent a corneal endothelial disorder orthe like due to a TGF-β signal, but it was surprising that an mTORinhibitor can also suppress overexpression of extracellular matrix incorneal endothelial cells. This suggests that an mTOR inhibitor cansimultaneously treat corneal endothelial disorders due to a TGF-β signaland overexpression of extracellular matrix in corneal endothelial cells.In particular, Fuchs' endothelial corneal dystrophy is a disease inwhich the density of corneal endothelial cells significantly decreasesdue to a TGF-β signal, and extracellular matrix is deposited in theDescemet's membrane, resulting in corneal guttae and hypertrophy of theDescemet's membrane. For this reason, suppression of the overexpressionof extracellular matrix means that therapy and prophylaxis of Fuchs'endothelial corneal dystrophy can be significantly improved, and iscapable of complete healing in some cases. It is also possible toimprove, treat, or prevent corneal guttae and hypertrophy of theDescemet's membrane, as well as other conditions associated withturbidity or deposition (irreversible turbidity in corneal stroma due toprotracted corneal edema or the like) that can occur due tooverproduction of extracellular matrix in corneal endothelial disorderssuch as Fuchs' endothelial corneal dystrophy.

In one embodiment, a corneal endothelial condition, disorder, or diseasedue to overexpression of extracellular matrix in corneal endothelialcells can be due to overexpression of fibronectin in corneal endothelialcells.

In one embodiment, a corneal endothelial condition, disorder, or diseasedue to overexpression of extracellular matrix in corneal endothelialcells is selected from the group consisting of Fuchs' endothelialcorneal dystrophy, guttae formation, hypertrophy of the Descemet'smembrane, hypertrophy of a cornea, turbidity, scar, turbidity in cornealstroma, corneal epithelial edema, corneal epithelial disorder,photophobia, and blurred vision.

In another aspect, the present invention provides a medicament for usein treating or preventing a corneal endothelial condition, disorder, ordisease due to a TGF-β signal and overexpression of extracellular matrixin corneal endothelial cells, comprising an mTOR inhibitor. An mTORinhibitor can simultaneously treat or prevent corneal endothelialdisorders due to a TGF-β signal and overexpression of extracellularmatrix in corneal endothelial cells.

In one embodiment, a corneal endothelial condition, disorder, or diseasedue to a TGF-β signal and overexpression of extracellular matrix incorneal endothelial cells is selected from the group consisting ofFuchs' endothelial corneal dystrophy, other endothelial cornealdystrophy, and a corneal endothelial disorder due to a drug, surgery,trauma, infection, uveitis, or the like.

In one embodiment, a corneal endothelial condition, disorder, or diseasedue to a TGF-β signal and overexpression of extracellular matrix incorneal endothelial cells comprises Fuchs' endothelial cornealdystrophy. Fuchs' endothelial corneal dystrophy is a disease in whichthe density of corneal endothelial cells significantly decreases due toa TGF-β signal and extracellular matrix is deposited in the Descemet'smembrane, resulting in a disorder such as corneal guttae and hypertrophyof the Descemet's membrane. For this reason, suppression of theoverexpression of extracellular matrix means that therapy cansignificantly improve Fuchs' endothelial corneal dystrophy, and completehealing in some cases. Improvement of a disorder or the like such ascorneal guttae and hypertrophy of the Descemet's membrane in Fuchs'endothelial corneal dystrophy using an mTOR inhibitor causes qualitativeimprovement in an ophthalmic disease and provides an unconventionaltherapeutic effect against a disease such as Fuchs' endothelial cornealdystrophy which was beyond saving.

In one embodiment, examples of utilization methods of the presentinvention include, but are not limited to, eye drops, as well asadministration methods such as injection into the anterior chamber,impregnation into a controlled-release agent, subconjunctival injection,and systemic administration (oral administration and intravenousinjection).

In one embodiment, the mTOR inhibitor used in the present invention canbe any type of mTOR inhibitor, as long as it is effective in treating orpreventing a given eye (e.g., corneal endothelial) condition, disorder,or disease. Specific mTOR inhibitors include at least one selected fromthe group consisting of rapamycin, temsirolimus, everolimus, PI-103,CC-223, INK128, AZD8055, KU 0063794, Voxtalisib (XL765, SAR245409),Ridaforolimus (Deforolimus, MK-8669), NVP-BEZ235, CZ415, Torkinib(PP242), Torin 1, Omipalisib (GSK2126458, GSK458), OSI-027, PF-04691502,Apitolisib (GDC-0980, RG7422), WYE-354, Vistusertib (AZD2014), Torin 2,Tacrolimus (FK506), GSK1059615, Gedatolisib (PF-05212384, PKI-587),WYE-125132 (WYE-132), BGT226 (NVP-BGT226), Palomid 529 (P529), PP121,WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388, Zotarolimus(ABT-578), and Chrysophanic Acid.

The above mTOR inhibitors may be used alone or in combination in themedicament of the present invention. The concentration of an mTORinhibitor used in the present invention can be appropriately changed inaccordance with the type of the mTOR inhibitor. For example, theconcentration can be, but is not limited to, at least about 0.0001 nM(nmol/L), at least about 0.001 nM, at least about 0.01 nM, at leastabout 0.1 nM, at least about 1 nM, at least about 10 nM, at least about100 nM, or at least about 1000 nM. The upper limit of the concentrationof an mTOR used in the present invention includes, but is not limitedto, about 100 μM (μmol/L), about 10 μM, about 1 μM, or about 0.5 μM.Examples of the concentration rage of an mTOR inhibitor used in thepresent invention include, but are not limited to, about 0.01 nM toabout 100 μM, about 0.1 nM to about 100 μM, about 1 nM to about 100 μM,about 10 nM to about 100 μM, about 100 nM to about 100 μM, about 1 μM toabout 100 μM, about 0.01 nM to about 10 μM, about 0.1 nM to about 10 μM,about 1 nM to about 10 μM, about 10 nM to about 10 μM, about 100 nM toabout 10 μM, about 1 μM to about 10 μM, about 0.01 nM to about 1 μM,about 0.1 nM to about 1 μM, about 1 nM to about 1 μM, about 10 nM toabout 1 μM, about 100 nM to about 1 μM, about 0.01 nM to about 100 nM,about 0.1 nM to about 100 nM, about 1 nM to about 100 nM, and about 10nM to about 100 nM. When two or more types of mTOR inhibitors are usedin combination, the concentration of each mTOR inhibitor can beappropriately changed.

In a preferred embodiment, an mTOR inhibitor is selected from the groupconsisting of, for example, rapamycin, temsirolimus, everolimus, andsalts thereof. Although not wishing to be bound by any theory, this isbecause it was found that treatment with mTOR inhibitors such asrapamycin, temsirolimus and everolimus exhibited a significantly bettertherapeutic result compared to other mTOR inhibitors, and results ofhealing especially a corneal endothelial disease or disorder associatedwith transforming growth factor-β2 (TGF-β2) such as Fuchs' endothelialdystrophy, or corneal endothelial disease or disorder associated withoverexpression of extracellular matrix (ECM) are significantly improved.In addition, this is because these mTOR inhibitors are already approvedby the FDA, PMDA and the like, and are expected to be able to beadministered as an ophthalmic medicament as a pharmaceutical producteven in view of aspects such as safety.

The above compounds may be used alone or in combination in themedicament of the present invention. The concentration of a compoundused in the present invention is about 0.01 nM to 100 μM (μmol/1), orabout 0.1 nM to 100 μm, generally about 1 nM to 100 μM, about 10 nM to100 μM, preferably about 0.1 to 30 μM, and more preferably about 1 to 10μM. The upper limits and lower limits thereof can be appropriately setin combination and when two or more types of compounds are used incombination, the concentration can be appropriately changed. Examples ofother concentration ranges include, but are not limited to, generallyabout 0.01 nM to 100 μM, about 0.1 nM to 100 μM, or about 0.001 to 100μM, preferably about 0.01 to 75 μM, about 0.05 to 50 μM, about 1 to 10μM, about 0.01 to 10 μM, about 0.05 to 10 μM, about 0.075 to 10 μM,about 0.1 to 10 μM, about 0.5 to 10 μM, about 0.75 to 10 μM, about 1.0to 10 μM, about 1.25 to 10 μM, about 1.5 to 10 μM, about 1.75 to 10 μM,about 2.0 to 10 μM, about 2.5 to 10 μM, about 3.0 to 10 μM, about 4.0 to10 μM, about 5.0 to 10 μM, about 6.0 to 10 μM, about 7.0 to 10 μM, about8.0 to 10 μM, about 9.0 to 10 μM, about 0.01 to 50 μM, about 0.05 to 5.0μM, about 0.075 to 5.0 μM, about 0.1 to 5.0 μM, about 0.5 to 5.0 μM,about 0.75 to 5.0 μM, about 1.0 to 5.0 μM, about 1.25 to 5.0 μM, about1.5 to 5.0 μM, about 1.75 to 5.0 μM, about 2.0 to 5.0 μM, about 2.5 to5.0 μM, about 3.0 to 5.0 μM, about 4.0 to 5.0 μM, about 0.01 to 3.0 μM,about 0.05 to 3.0 μM, about 0.075 to 3.0 μM, about 0.1 to 3.0 μM, about0.5 to 3.0 μM, about 0.75 to 3.0 μM, about 1.0 to 3.0 μM, about 1.25 to3.0 μM, about 1.5 to 3.0 μM, about 1.75 to 3.0 μM, about 2.0 to 3.0 μM,about 0.01 to 1.0 μM, about 0.05 to 1.0 μM, about 0.075 to 1.0 μM, about0.1 to 1.0 μM, about 0.5 to 1.0 μM, about 0.75 to 1.0 μM, about 0.09 to35 μM, about 0.09 to 3.2 μM, more preferably about 0.05 to 1.0 μM, about0.075 to 1.0 μM, about 0.1 to 1.0 μM, about 0.5 to 1.0 μM, about 0.75 to1.0 μM.

When used as an eye drop, the formulation concentration can bedetermined using about 1 to 10000-fold, preferably about 100 to10000-fold such as about 1000-fold of the above effective concentrationas a reference while considering dilution with tear fluid or the likeand paying attention to toxicity. It is also possible to set a higherconcentration. For example, the concentration is about 0.01 μM (μMol/l)to 1000 mM (mmol/l), about 0.1 μM to 100 mM, about 1 μM to 100 mM, about10 μM to 100 mM, or about 0.1 μM to 30 mM, about 1 μM to 30 mM, morepreferably about 1 μM to 10 mM, about 10 μM to 10 mM, about 100 μM to 10mM, about 10 μM to 100 mM, about 100 μM to 100 mM, or can be about ¥ mMto 10 mM, about 1 mM to 100 mM. The upper limits and lower limitsthereof can be appropriately set in combination and when two or moretypes of compounds are used in combination, the concentration can beappropriately changed.

In another embodiment, an mTOR inhibitor is rapamycin. The concentrationof the rapamycin to be used is at least about 0.1 nM, at least about 1nM, at least about 10 nM, preferably about 100 nM. In a preferableembodiment, rapamycin is used as an eye drop and the concentration ofthe rapamycin to be used upon doing so is at least about 0.1 mM, atleast about 1 mM, at least about 10 mM, preferably at least about 100mM. Saturation amount or 1000 mM is exemplified as the upper limit ofthe concentration.

In another embodiment, an mTOR inhibitor is temsirolimus. Theconcentration of the temsirolimus to be used is at least about 0.01 nM,at least about 0.1 nM, preferably about 1 nM, preferably about 10 nM,more preferably about 100 nM, more preferably about 1 μM, morepreferably about 10 μM. Temsirolimus is used as an eye drop and theconcentration of the temsirolimus to be used upon doing so is at leastabout 0.01 mM, at least about 0.1 mM, at least about 1 mM, preferably atleast about 10 mM. Saturation amount or 1000 mM is exemplified as theupper limit of the concentration. In another embodiment, an mTORinhibitor is everolimus. The concentration of the everolimus to be usedis at least about 0.1 nM, at least about 1 nM, at least about 10 nM,preferably about 100 nM. Everolimus is used as an eye drop and theconcentration of the everolimus to be used upon doing so is at leastabout 0.1 mM, at least about 1 mM, at least about 10 mM, preferably atleast about 100 mM. Saturation amount or 1000 mM is exemplified as theupper limit of the concentration.

In one embodiment, a therapeutic or prophylactic medicament of thepresent invention can be targeted for any animal with a cornealendothelium, such as mammals. Such a medicament is preferably intendedfor treating or preventing a primate corneal endothelium. The subject oftherapy or prophylaxis is preferably a human corneal endothelium.

In still another embodiment, an mTOR inhibitor may be an mTOR geneexpression suppressing substance. Examples of an mTOR gene expressionsuppressing substance include, but are not limited to, siRNA, antisensenucleic acid, or ribozyme.

In a specific embodiment, an mTOR inhibitor is an siRNA against an mTORgene. Typical examples of siRNA used in the present invention include,but are not limited to:

a sense strand set forth in CAUUCGCAUUCAGUCCAUAtt (SEQ ID NO: 1); and

an antisense strand set forth in UAUGGACUGAAUGCGAAUGat (SEQ ID NO: 2).

Any sequence is acceptable as long as there is an antisense effect or asense RNAi effect against an mTOR gene. 1 to 3 bases of nucleotides maybe deleted, substituted, inserted and/or added in these sense strand andantisense strands.

In another aspect, the present invention provides a method of treatingor preventing an eye (e.g., corneal endothelial) condition, disorder, ordisease (corneal endothelial condition, disorder, or disease due to aTGF-β signal and/or overexpression of extracellular matrix in cornealendothelial cells), comprising administering an effective amount of anmTOR inhibitor to a subject in need thereof.

As used herein, a “subject” refers to a target of administration(transplant) of a therapeutic or prophylactic medicament or method ofthe present invention. Examples of subjects include mammals (e.g.,human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkeyand the like), but primates are preferable and humans are especiallypreferable.

The effective amount of the medicament of the present invention, whichis effective in treating a specific disease, disorder, or condition, canvary depending on the properties of a disorder or condition, but theeffective amount can be determined by those skilled in the art withstandard clinical techniques based on the descriptions in the presentspecification. It is also possible to use an in vitro assay to assist inidentifying the optimal range of dosage as needed. Since an accuratedose to be used in a formulation can vary depending on the route ofadministration and the severity of a disease or disorder, the doseshould be determined in accordance with the judgment of a physician andthe condition of each patient. However, the dosage, while notparticularly limited, may be, for example, 0.001, 1, 5, 10, 15, 100, or1000 mg/kg body weight or a value between any two such values per dose.The interval of administration, while not particularly limited, may befor example one or two doses for every 1, 7, 14, 21, or 28 days, or oneor two doses for a number of days between any two such values. Thedosage, number of doses, administration interval, and administrationmethod may be appropriately selected depending on the age or body weightof a patient, condition, dosage form, target organ, or the like. Forexample, the present invention can be used as an eye drop. Themedicament of the present invention can also be injected into theanterior chamber. A therapeutic drug preferably comprises atherapeutically effective amount or an effective amount of activeingredients at which a desired action is exerted. It may be determinedthat there is a therapeutic effect when a therapeutic markersignificantly decreases after administration. The effective amount canbe estimated from a dose-response curve obtained from an in vitro oranimal model testing system.

<Preservation and Growth of Corneal Endothelial Cells>

In another aspect, the present invention provides a composition forpreserving corneal endothelial cells, comprising an mTOR inhibitor. Thepresent invention also provides a method for preserving cornealendothelial cells, encompassing contacting an effective amount of anmTOR inhibitor with corneal endothelial cells. It is understood that anembodiment of an mTOR inhibitor or the like used for preservation ofcorneal endothelial cells can use any embodiment described in the<Medicament> in the present specification. Contact to cornealendothelial cells can be performed in vivo, ex vivo, or in vitro, andcan be used in the manufacturing of a cell formulation.

In another aspect, the present invention provides a composition forgrowing or promoting the growth of corneal endothelial cells, comprisingan mTOR inhibitor. The present invention also provides a method forgrowing or promoting the growth of corneal endothelial cells,encompassing contacting an effective amount of an mTOR inhibitor withcorneal endothelial cells. It is understood that an embodiment of anmTOR inhibitor or the like used for growing or promoting the growth ofcorneal endothelial cells can use any embodiment described in the<Medicament> in the present specification. Contact to cornealendothelial cells can be performed in vivo, ex vivo, or in vitro, andcan be used in the manufacturing of a cell formulation.

Reference literature such as scientific literature, patents, and patentapplications cited herein is incorporated herein by reference to thesame extent that the entirety of each document is specificallydescribed.

The present invention has been explained while showing preferredembodiments to facilitate understanding. The present invention isexplained hereinafter based on Examples. The above explanation and thefollowing Examples are not provided to limit the present invention, butfor the sole purpose of exemplification. Thus, the scope of the presentinvention is not limited to the embodiments and Examples that arespecifically disclosed herein and is limited only by the scope ofclaims.

EXAMPLES

Hereinafter, examples of the present invention are described. Biologicalsamples or the like, where applicable, were handled in compliance withthe standards enacted by the Ministry of Health, Labour and Welfare,Ministry of Education, Culture, Sports, Science and Technology, or thelike and, where applicable, based on the Helsinki Declaration or ethicalcodes prepared based thereon. For the donation of eyes used for thestudy, consent was obtained from close relatives of all deceased donors.The present study was approved by the ethics committee or acorresponding body of the University of Erlangen-Nuremberg (Germany) andSightLife™ (Seattle, Wash.) eye bank.

Preparation Example: Production of Fuchs' Endothelial Corneal DystrophyPatient Derived Immortalized Corneal Endothelial Cell Line (iFECD) Model

In the present example, an immortalized corneal endothelial cell line(iFECD) was made from corneal endothelial cells from Fuchs' endothelialcorneal dystrophy patients.

(Culture Method)

Corneal endothelial cells were mechanically peeled off with a basalmembrane from a cornea for research purchased from the Seattle Eye Bank.After using collagenase to detach and collect the corneal endothelialcell from the basal membrane, the cells were subjected to primaryculture. For the medium, Opti-MEM I Reduced-Serum Medium, Liquid(INVITROGEN catalog number: 31985-070), to which 8% FBS (BIOWEST,catalog number: S1820-500), 200 mg/mL of CaCl₂.2H₂O (SIGMA catalognumber: C7902-500G), 0.08% of chondroitin sulfate (SIGMA catalog number:C9819-5G), 20 μg/mL of ascorbic acid (SIGMA catalog number: A4544-25G),50 μg/mL of gentamicin (INVITROGEN catalog number: 15710-064) and 5ng/mL of EGF (INVITROGEN catalog number: PHG0311) were added, andconditioned for a 3T3 feeder cell was used as a basal medium. Further,the cells were cultured in a basal medium to which SB431542 (1 μmol/L)and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole<4-[4-(4-fluorphenyl)-2-(4-methylsulfinylphenyl)-1H-imidazole-5-yl]pyridine)(1 μmol/L) were added (also referred to as “SB203580+SB431542+3T3conditioned medium” herein).

(Method of Acquisition)

Corneal endothelial cells were obtained with approval from an ethicscommittee and written consent from 3 human patients who suffered frombullous keratopathy according to a clinical diagnosis of Fuchs'endothelial corneal dystrophy and underwent corneal endothelialtransplant (Descemet's Membrane Endothelial Keratoplasty=DMEK). ForDMEK, pathological corneal endothelial cells were mechanically peeledoff with the basal membrane, i.e., the Descemet's membrane, and immersedin a cornea preservation solution Optisol-GS (Bausch & Lomb).Collagenase treatment was then applied to enzymatically collect thecorneal endothelial cells, and the cells were cultured with aSB203580+SB431542+3T3 conditioned medium. For cultured cornealendothelial cells from a Fuchs' endothelial corneal dystrophy patient,SV40 large T antigen and hTERT gene were amplified by PCR and introducedinto a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc). Thelentiviral vector was then used to infect 293T cells (RCB2202; RikenBioresource Center, Ibaraki, Japan) with a transfection reagent (FugeneHD; Promega Corp., Madison, Wis.) and three types of helper plasmids(pLP1, pLP2, pLP/VSVG; Life Technologies Inc.). Culture supernatantcomprising viruses was collected after 48 hours from the infection. 5μg/ml of polybrene was used and added to a culture solution of culturedcorneal endothelial cells from a Fuchs' endothelial corneal dystrophypatient, and SV40 large T antigen and hTERT gene were introduced. Imagesof immortalized corneal endothelial cell line (iFECD) from Fuchs'endothelial corneal dystrophy patients from a phase differencemicroscope were studied. Cultured corneal endothelial cells from aresearch cornea imported from the Seattle Eye Bank were immortalized bythe same method to make an immortalized cell line of normal cornealendothelial cells (iHCEC) as a control. When images of the immortalizedcorneal endothelial cell line (iFECD) and the immortalized cornealendothelial cell line from a healthy donor (iHCEC) from a phasedifference microscope are studied, both iHCEC and iFECD have a layer ofpolygonal form as in normal corneal endothelial cells. IHCEC and iFECDwere maintained and cultured with Dulbecco's Modified Eagle Medium(DMEM)+10% fetal bovine serum (FBS).

Example 1: Suppression Effect of Rapamycin on Cell Damage Induced byTGF-β2

This Example demonstrated the effect of rapamycin, which is a typicalexample of an mTOR inhibitor, on cell damage.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 1×10⁵ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (DMEM+10% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. Rapamycinwas added to culture the cells for 24 hours. (DMEM+2% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. A mediumcontaining 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911) andrapamycin was added to culture the cells for 24 hours. (DMEM+2% FBS+1%P/S was used as the medium). After 24 hours, the cell morphology andapoptosis were observed under a phase contrast microscope.

(Results)

(Rapamycin Suppresses Cell Damage Induced by TGF-β2)

FIG. 1 shows the results. When iFECDs were stimulated with RecombinantHuman TGF-β2 in the absence of rapamycin, cells were found to besignificantly damaged. On the other hand, it was observed that damage tocorneal endothelial cells was suppressed when pretreated with rapamycin.Therefore, rapamycin was found to suppress cell damage induced byRecombinant Human TGF-β2.

Example 2: Suppression Effect of Rapamycin on Caspase Activity Inducedby TGF-β2

This Example demonstrated the effect of rapamycin, which is a typicalexample of an mTOR inhibitor, on caspase activity.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 1×10⁵ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (DMEM+10% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. Rapamycinwas added to culture the cells for 24 hours. (DMEM+2% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. A mediumcontaining 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911) andrapamycin was added to culture the cells for 24 hours. (DMEM+2% FBS+1%P/S was used as the medium). After 24 hours, the cell morphology andapoptosis were observed under a phase contrast microscope.

After observation, western blot was performed on proteins by thefollowing procedure.

1) Protein Collection

The medium was collected on ice to collect free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

8 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A rabbit anti-Caspase 3 antibody (CellSignaling, 9662), rabbit anti-PARP antibody (Cell Signaling, 9542), andmouse anti-GAPDH antibody (MBL, M171-3) were used as the primaryantibodies. A peroxidase-labeled anti-rabbit antibody and anti-mouseantibody (GE Healthcare Biosciences, NA931V, NA934V) were used as thesecondary antibodies. For the primary antibodies, rabbit anti-Caspase 3antibody: 1000-fold dilution, rabbit anti-PARP antibody: 2000-folddilution, and mouse anti-GAPDH antibody: 5000-fold dilution, while thesecondary antibody was diluted 5000-fold. Chemi Lumi ONE Ultra (NacalaiTesque, 11644-40) was used for detection. The detected band strength wasanalyzed with a lumino image analyzer LAS-4000 mini (Fuji Film) andImageQuant™ software (GE Healthcare).

(Results)

(Rapamycin Suppresses Caspase Activity Induced by TGF-β2)

The results of western blot of caspase are shown in FIG. 2. When iFECDswere stimulated with Recombinant Human TGF-β2 in the absence ofrapamycin, cleaved caspase 3 (about 17 kDa), which is an active form,was observed. On the other hand, activated form of cleaved caspase 3 washardly observed in the rapamycin supplemented group. Therefore, caspaseactivation by Recombinant Human TGF-β2 was found to be suppressed byrapamycin in the analysis. An mTOR is not related to caspase signal andit is not known that an mTOR inhibitor suppresses caspase activity.Thus, it was surprising that rapamycin was able to suppress caspaseactivity.

Example 3: Suppression Effect of Rapamycin on Phosphorylation Activityof S6K Induced by Recombinant Human TGF-β2

This Example demonstrated the effect of rapamycin, which is a typicalexample of an mTOR inhibitor, on phosphorylation activity of S6K inducedby Recombinant Human TGF-β2.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 7×10⁴ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (DMEM+10% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. Rapamycinwas added to culture the cells for 24 hours. (DMEM+2% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. A mediumcontaining 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911) andrapamycin was added to culture the cells for 24 hours. (DMEM+2% FBS+1%P/S was used as the medium). After 24 hours, the cell morphology andapoptosis were observed under a phase contrast microscope.

After observation, western blot was performed on proteins by thefollowing procedure.

1) Protein Collection

The medium was collected on ice to collect free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

8 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A rabbit anti-Akt1 antibody (CellSignaling, 2938), mouse anti-Phospho-Akt antibody (Cell Signaling,4051), rabbit anti-S6K antibody (Cell Signaling, 9202), rabbitanti-Phospho-S6K antibody (Cell Signaling, 9204) and mouse anti-GAPDHantibody (MBL, M171-3) were used as the primary antibodies. Aperoxidase-labeled anti-rabbit antibody and anti-mouse antibody (GEHealthcare Biosciences, NA931V, NA934V) were used as the secondaryantibodies. For the primary antibodies, rabbit anti-Akt antibody:1000-fold dilution, mouse anti-Phospho-Akt antibody: 1000-fold dilution,rabbit anti-S6K antibody: 1000-fold dilution, rabbit anti-Phospho-S6Kantibody: 1000-fold dilution, and mouse anti-GAPDH antibody: 5000-folddilution, while the secondary antibody was diluted 5000-fold. Chemi LumiONE Ultra (Nacalai Tesque, 11644-40) was used for detection. Thedetected band strength was analyzed with a lumino image analyzerLAS-4000 mini (Fuji Film) and ImageQuant™ software (GE Healthcare).

(Results)

(Rapamycin Suppresses Phosphorylation Activity of S6K induced by TGF-β2)

The results are shown in FIG. 3. When iFECDs were stimulated withRecombinant Human TGF-β2 in the absence of rapamycin, phosphorylationactivity of Akt was found. On the other hand, phosphorylation activityof S6K was suppressed in the rapamycin supplemented group. Rapamycin wasfound to have mTOR signal pathway inhibitory action by western blotanalysis in rapamycin. Akt is present upstream of mTOR and S6K ispresent downstream of mTOR. Thus, in view of the fact that the upstreamAkt was phosphorylated while phosphorylation of the downstream S6K wassuppressed by the mTOR inhibitor, rapamycin was found to be inhibitingan mTOR pathway.

Example 4: Suppression Effect of Rapamycin on Phosphorylation Activityof Smad2/3 Induced by TGF-β2

This Example demonstrated suppression effect of rapamycin, which is atypical example of an mTOR inhibitor, on phosphorylation activity ofSmad2/3 induced by TGF-β2.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 8×10⁴ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (DMEM+10% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. Rapamycinwas added to culture the cells for 24 hours. (DMEM+2% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. A mediumcontaining 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911) andrapamycin was added to culture the cells for 24 hours. (DMEM+2% FBS+1%P/S was used as the medium). After 24 hours, the cell morphology andapoptosis were observed under a phase contrast microscope.

After observation, western blot was performed on proteins by thefollowing procedure.

1) Protein Collection

The Medium was Collected on Ice to Collect Free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

8 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A rabbit anti-Smad2 antibody (CellSignaling, 5339), mouse anti-Phospho-Smad2 antibody (Cell Signaling,3108), rabbit anti-Smad3 antibody (Cell Signaling, 9523), rabbitanti-Phospho-Smad3 antibody (Cell Signaling, 9520) and mouse anti-GAPDHantibody (MBL, M171-3) were used as the primary antibodies. Aperoxidase-labeled anti-rabbit antibody and anti-mouse antibody (GEHealthcare Biosciences, NA931V, NA934V) were used as the secondaryantibodies. For the primary antibodies, rabbit anti-Smad2 antibody:1000-fold dilution, mouse anti-Phospho-Smad2 antibody: 1000-folddilution, rabbit anti-Smad3 antibody: 1000-fold dilution, rabbitanti-Phospho-Smad3 antibody: 1000-fold dilution, and mouse anti-GAPDHantibody: 5000-fold dilution, while the secondary antibody was diluted5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used fordetection. The detected band strength was analyzed with a lumino imageanalyzer LAS-4000 mini (Fuji Film) and ImageQuant™ software (GEHealthcare).

(Results)

(Rapamycin does not Suppress Phosphorylation Activity of Smad2/3 Inducedby TGF-β2)

The results are shown in FIG. 4. When stimulated with Recombinant HumanTGF-β2 in the absence of rapamycin, phosphorylation activities of Smad2and Smad3 were found. Thus, phosphorylation activity of Smad2/3 inducedby Recombinant Human TGF-β2 was found not to be suppressed by rapamycinin western blot analysis. It was revealed that cell damage suppressionaction of rapamycin is not due to inhibition of a TGF-β signal since theaction of TGF-β is understood to be transmitted by a phosphorylationpathway of Smad. This suggests that rapamycin suppresses a cornealendothelial disorder and the like due to a TGF-β signal without directlyinhibiting the TGF-β signal, which was an unexpected result.

Example 5: Suppression Effect of Rapamycin on Production of FibronectinInduced by TGF-β2

This Example demonstrated suppression effect of rapamycin, which is atypical example of an mTOR inhibitor, on the production of fibronectininduced by TGF-β2.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 6-well plate at a ratio of 2×10⁵ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (DMEM+10% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. Rapamycinwas added to culture the cells for 24 hours. (DMEM+2% FBS+1% P/S wasused as the medium). After 24 hours, the medium was removed. A mediumcontaining 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911) andrapamycin was added to culture the cells for 24 hours. (DMEM+2% FBS+1%P/S was used as the medium). After 24 hours, the cell morphology andapoptosis were observed under a phase contrast microscope.

After observation, western blot was performed on proteins by thefollowing procedure.

1) Protein Collection

The medium was collected on ice to collect free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

5 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A mouse anti-Fibronectin antibody (BDBioscience, 610077), and mouse anti-GAPDH antibody (MBL, M171-3) wereused as the primary antibodies. A peroxidase-labeled anti-rabbitantibody and anti-mouse antibody (GE Healthcare Biosciences, NA931V,NA934V) were used as the secondary antibodies. For the mouseanti-Fibronectin antibody: 15000-fold dilution and mouse anti-GAPDHantibody: 5000-fold dilution, while the secondary antibody was diluted5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used fordetection. The detected band strength was analyzed with a lumino imageanalyzer LAS-4000 mini (Fuji Film) and ImageQuant™ software (GEHealthcare).

(Results)

(Rapamycin Suppresses Production of Fibronectin Induced by TGF-β2)

The results are shown in FIG. 5. When stimulated with Recombinant HumanTGF-β2 in the absence of rapamycin, production of fibronectin wasobserved in iFECD. On the other hand, production of fibronectin washardly observed in the rapamycin supplemented group. Therefore, theamount of expression of fibronectin induced by Recombinant Human TGF-β2was found to be suppressed by rapamycin in western blot analysis. It wasnot known that an mTOR inhibitor is involved in the production ofextracellular matrix such as fibronectin. Thus, it was unexpected thatan mTOR is able to suppress extracellular matrix production.

In Fuchs' endothelial corneal dystrophy, overproduction of extracellularmatrix such as fibronectin and deposition thereof on the Descemet'smembrane result in a disorder such as hypertrophy of the Descemet'smembrane or guttae formation. Such a disorder commonly starts developingin the 30s and 40s in Fuchs' endothelial corneal dystrophy patients, andprogresses throughout the patient's life. Progression results in visualimpairment such as blurred vision, halo, glare, or reduced vision. Whilecorneal endothelial cells are continually damaged in Fuchs' endothelialcorneal dystrophy, the transparency of the corneal is maintained by theremaining corneal endothelia compensating for the pumping function untilthe corneal endothelial cell density is below about 1000 cells/mm². Ifthe density is below about 1000 cells/mm², infiltration of anterioraqueous humor into the cornea leads to corneal edema, resulting invisual impairment (FIG. 18). In this manner, Fuchs' endothelial cornealdystrophy patients suffer from a visual function disorder due to mainlytwo causes, i.e., overproduction of extracellular matrix and cornealendothelial cell death. The caspase inhibitor in the present inventionhave a role in both suppression of extracellular matrix production andsuppression of corneal endothelial cell death to be especially useful inthe therapy of Fuchs' endothelial corneal dystrophy.

Example 6: mTOR siRNA Suppression Effect on mTOR Expression andPhosphorylation of S6K

This Example demonstrated the suppression effect of mTOR siRNA, which isanother typical example of an mTOR inhibitor, on mTOR expression andphosphorylation of S6K.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 3×10⁴ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (medium: DMEM+10% FBS+1%P/S). After 24 hours, the medium was removed. Lipofectamine (invitrogen,92008) and 3 pmol of mTOR siRNA (Ambion, AS026M0L) were added to culturethe cells for 24 hours (medium: OptiMEM-I (invitrogen, 31985-088)). ThemTOR siRNA that was used has a sense strand set forth in SEQ ID NO: 1and an antisense strand set forth in SEQ ID NO: 2. After 24 hours, themedium was removed to culture the cells for 72 hours at 37° C. (5% CO₂)(medium: DMEM+10% FBS+1% P/S). After 72 hours, the medium was removed.Media containing 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911)were added to culture the cells for 24 hours (medium: DMEM+2% FBS+1%P/S). After 24 hours, the cell morphology and apoptosis were observedunder a phase difference microscope.

After observation, amplification of a base sequence of cDNA was carriedout by PCR method with the following procedure.

1) Total RNA Collection

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 350 μl of Buffer RLTLysis buffer of RNeasy miniKit (QIAGEN, M610A), and were eluted. Thecells were supplemented with 350 μl of 70% EtOH and moved to RNeas minispin column (QIAGEN) to then be centrifuged for 15 seconds at 10000 rpmto discard Flow-through. Furthermore, 30 μl of RNeasy free water(QIAGEN) was supplemented to RNeasy mini spin column to then carry outcentrifugation for 1 minute at 10000 rpm to extract total RNA.

2) cDNA Synthesis

Each of the extracted total RNA 450 ng, Rever Tra Ace (TOYOBO), 10 mMdNTP Mixture (TOYOBO), 5×RT Buffer (TOYOBO) and random primer(invitrogen) were added to synthesize an complementary DNA using T3000Thermocycler (biometra). The reaction condition was to carry outannealing reaction for 10 minutes at 30° C., reverse transcriptionreaction for 60 minutes at 42° C. and thermal denaturation reaction for5 minutes at 99° C.

3) PCR Method

1 μl of the synthesized complementary DNA, 5 μl of 2×GO Taq GreenMasterMix (Promega), 1 μl of Forward custom primer (invitrogen) of mTOR, 1 μlof Reverse custom primer (invitrogen) and 2 μl of H₂O were mixed toamplify the base sequence of the complementary DNA using T3000Thermocycler (biometra). The reaction condition was to carry out initialthermal denaturation reaction for 2 minutes at 94° C., followed by 26cycles of thermal denaturation reaction for 30 seconds at 95° C.,annealing reaction for 20 seconds at 53° C. and elongation reaction for25 seconds at 72° C. Furthermore, elongation reaction was carried outfor 5 minutes at 72° C. After the amplification, detection was carriedout by UV irradiation with Amersham™ Imager 600 (GE Healthcare Japan)and electrophoresis using agarose gel.

Furthermore, after observation, western blot was performed on proteinsby the following procedure.

1) Protein Collection

The medium was collected on ice to collect free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

5 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A rabbit anti-mTOR antibody (CellSignaling, 2972), rabbit anti-S6K antibody (Cell Signaling, 9202),rabbit anti-Phospho-S6K antibody (CellSignaling, 9204), and mouseanti-GAPDH antibody (MBL, M171-3) were used as the primary antibodies. Aperoxidase-labeled anti-rabbit antibody and anti-mouse antibody (GEHealthcare Biosciences, NA931V, NA934V) were used as the secondaryantibodies. For the primary antibodies, rabbit anti-mTOR antibody:1000-fold dilution, rabbit anti-S6K antibody: 1000-fold dilution, rabbitanti-Phospho-S6K antibody: 1000-fold dilution and mouse anti-GAPDHantibody: 5000-fold dilution, while the secondary antibody was diluted5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used fordetection. The detected band strength was analyzed with a lumino imageanalyzer LAS-4000 mini (Fuji Film) and ImageQuant™ software (GEHealthcare).

(Results)

(mTOR siRNA Suppresses mTOR Expression and Phosphorylation of S6K)

The results are shown in FIG. 6. When RNAi is carried out against anmTOR in iFECD, mTOR expression suppression was found in both RNA leveland protein level. In addition, phosphorylation of S6K was found to besuppressed in the protein level by mTOR siRNA. Thus, mTOR expression andphosphorylation of S6K were found to be suppressed by mTOR siRNA in PCRand western blot analyses.

Example 7: Suppression Effect of mTOR siRNA on Cell Damage Induced byTGF-β2

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 3×10⁴ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (medium: DMEM+10% FBS+1%P/S). After 24 hours, the medium was removed. Lipofectamine (invitrogen,92008) and 3 pmol of mTOR siRNA (Ambion, AS026M0L) were added to culturethe cells for 24 hours (medium: OptiMEM-I (invitrogen, 31985-088)). ThemTOR siRNA that was used has a sense strand set forth in SEQ ID NO: 1and an antisense strand set forth in SEQ ID NO: 2. After 24 hours, themedium was removed to culture the cells for 72 hours at 37° C. (5% CO₂)(medium: DMEM+10% FBS+1% P/S). After 72 hours, the medium was removed.Media containing 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911)were added to culture the cells for 24 hours (medium: DMEM+2% FBS+1%P/S). After 24 hours, the cell morphology and apoptosis were observedunder a phase difference microscope.

(Results)

(mTOR siRNA Suppresses Cell Damage Induced by TGF-β2)

The results are shown in FIG. 7. When iFECDs were stimulated withRecombinant Human TGF-β2 without using mTOR siRNA, cells were found tobe significantly damaged. On the other hand, it was observed that damageto corneal endothelial cells was suppressed when mTOR siRNA was used.Therefore, mTOR expression suppression was found to suppress cell damageinduced by TGF-β2.

Example 8: Suppression Effect of mTOR siRNA on Caspase ActivationInduced by TGF-β2

This Example demonstrated the suppression effect of mTOR siRNA, which isanother typical example of an mTOR inhibitor, on caspase activationinduced by TGF-β2.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 3×10⁴ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (medium: DMEM+10% FBS+1%P/S). After 24 hours, the medium was removed. Lipofectamine (invitrogen,92008) and 3 pmol of mTOR siRNA (Ambion, AS026M0L) were added to culturethe cells for 24 hours (medium: OptiMEM-I (invitrogen, 31985-088)). ThemTOR siRNA that was used has a sense strand set forth in SEQ ID NO: 1and an antisense strand set forth in SEQ ID NO: 2. After 24 hours, themedium was removed to culture the cells for 72 hours at 37° C. (5% CO₂)(medium: DMEM+10% FBS+1% P/S). After 72 hours, the medium was removed.Media containing 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911)were added to culture the cells for 24 hours (medium: DMEM+2% FBS+1%P/S). After 24 hours, the cell morphology and apoptosis were observedunder a phase difference microscope.

Furthermore, after observation, western blot was performed on proteinsby the following procedure.

1) Protein Collection

The medium was collected on ice to collect free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

6 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A rabbit anti-Caspase 3 antibody (CellSignaling, 9662), rabbit anti-PARP antibody (Cell Signaling, 9542), andmouse anti-GAPDH antibody (MBL, M171-3) were used as the primaryantibodies. A peroxidase-labeled anti-rabbit antibody and anti-mouseantibody (GE Healthcare Biosciences, NA931V, NA934V) were used as thesecondary antibodies. For the primary antibodies, rabbit anti-Caspase 3antibody: 1000-fold dilution, rabbit anti-PARP antibody: 2000-folddilution and mouse anti-GAPDH antibody: 5000-fold dilution, while thesecondary antibody was diluted 5000-fold. Chemi Lumi ONE Ultra (NacalaiTesque, 11644-40) was used for detection. The detected band strength wasanalyzed with a lumino image analyzer LAS-4000 mini (Fuji Film) andImageQuant™ software (GE Healthcare).

(Results)

(mTOR siRNA Suppresses Caspase Activation Induced by TGF-β2)

The results are shown in FIG. 8. When iFECDs were stimulated withRecombinant Human TGF-β2 without using mTOR siRNA, about 17 kDa ofcleaved caspase-3, which is an active form, was observed in iFECD. Onthe other hand, activated form of cleaved caspase-3 was hardly observedin the mTOR siRNA supplemented group. Thus, caspase activation inducedby TGF-β2 was demonstrated to be suppressed by western blot analysis inmTOR signal suppression.

Example 9: Suppression Effect of mTOR siRNA on Production of FibronectinInduced by TGF-β2

This Example demonstrated the suppression effect of mTOR siRNA, which isanother typical example of an mTOR inhibitor, on production offibronectin induced by TGF-β2

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

iFECDs were seeded on a 12-well plate at a ratio of 3×10⁴ cells per welland cultured for 24 hours at 37° C. (5% CO₂) (medium: DMEM+10% FBS+1%P/S). After 24 hours, the medium was removed. Lipofectamine (invitrogen,92008) and 3 pmol of mTOR siRNA (Ambion, AS026M0L) were added to culturethe cells for 24 hours (medium: OptiMEM-I (invitrogen, 31985-088)). ThemTOR siRNA that was used has a sense strand set forth in SEQ ID NO: 1and an antisense strand set forth in SEQ ID NO: 2. After 24 hours, themedium was removed to culture the cells for 48 hours at 37° C. (5% CO₂)(medium: DMEM+10% FBS+1% P/S). After 48 hours, the medium was removed.Media containing 10 ng/ml of Recombinant Human TGF-β2 (Wako, 200-19911)were added to culture the cells for 24 hours (medium: DMEM+2% FBS+1%P/S). After 24 hours, the cell morphology and apoptosis were observedunder a phase difference microscope.

Furthermore, after observation, western blot was performed on proteinsby the following procedure.

1) Protein Collection

The medium was collected on ice to collect free and dead cells. Thesolution from washing the cells twice with 1×PBS (−) was also collected.800 g was centrifuged at 4° C. for 12 minutes. The supernatant wasdiscarded to obtain precipitates. The washed cells were supplementedwith a protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice to extractproteins. The precipitates from centrifuging the free and dead cellswere also subsequently suspended together for extraction. The collectedsolution was pulverized three times for 30 seconds in cold water with asonication device (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at4° C. at 15000 rpm to collect the supernatant of proteins.

2) Western Blot

7 μg of the extracted protein was separated by SDS-PAGE and transferredonto a nitrocellulose membrane. A mouse anti-Fibronectin antibody(BDBioscience, 610077) and mouse anti-GAPDH antibody (MBL, M171-3) wereused as the primary antibodies. A peroxidase-labeled anti-rabbitantibody and anti-mouse antibody (GE Healthcare Biosciences, NA931V,NA934V) were used as the secondary antibodies. For the primaryantibodies, rabbit anti-Caspase 3 antibody: 1000-fold dilution, rabbitanti-PARP antibody: 2000-fold dilution, and mouse anti-GAPDH antibody:5000-fold dilution, while the secondary antibody was diluted 5000-fold.Chemi Lumi ONE Ultra (Nicolai tissue, 11644-40) was used for detection.The detected band strength was analyzed with a lumino image analyzerLAS-4000 mini (Fuji Film) and ImageQuant™ software (GE Healthcare).

(Results)

(mTOR siRNA Suppresses Production of Fibronectin Induced by RecombinantHuman TGF-β2)

The results are shown in FIG. 9. When iFECDs were stimulated withRecombinant Human TGF-β2 without using mTOR siRNA, production offibronectin was observed in iFECD. On the other hand, production offibronectin was hardly observed in the mTOR siRNA supplemented group.Thus, expression of fibronectin was found to be suppressed by westernblot analysis in mTOR siRNA.

Example 10: Suppression Effect of Each mTOR Inhibitor on CaspaseActivity Induced by TGF-β2

This Example demonstrated suppression effect of each type of mTORinhibitor on the caspase activity induced by TGF-β2.

(Materials and Methods)

The medium was removed from a culture dish in which iFECDs were beingcultured, and the cells were supplemented with 1×PBS (−) that waspreheated to 37° C., and were washed. This was repeated twice. The cellswere supplemented again with 1×PBS (−) and incubated for 3 minutes at37° C. (5% CO₂). After removing the PBS (−), the cells were supplementedwith 0.05% Trypsin-EDTA (Nacalai Tesque, 32778-34) and incubated for 5minutes at 37° C. (5% CO₂). The cells were then suspended in a medium,and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (NacalaiTesque, 08456-36)+10% FBS (Biowest, S1820-500)+1% P/S (Nacalai Tesque,26252-94) was used as the medium.

Corneal endothelial cells from immortalized FECD patients (lot:iFECD3-5) were seeded on a 96-well plate at a ratio of 4×10³ cells perwell and cultured for 24 hours at 37° C. (5% CO₂) (medium: DMEM+10%FBS+1% P/S). After 24 hours, the medium was removed. Each mTOR inhibitorwas added to culture the cells for 24 hours (medium: DMEM+2% FBS+1%P/S). After 24 hours, the medium was removed. Media containing 10 ng/mlof Recombinant Human TGF-β2 (Wako, 200-19911) and each mTOR inhibitorwere added to culture the cells for 24 hours (medium: DMEM+2% FBS+1%P/S). After 24 hours, the cell morphology and apoptosis were observedunder a phase difference microscope.

After observation, measurement of caspase 3/7 activity by Caspase-Glo®3/7 Assay was carried out with the following procedure.

The medium was discarded to achieve 50 μl per well. 50 μl/well ofCaspase Glo® 3/7 Assay Reagent (mixture of Caspase-Glo® 3/7 Assay Bufferand Caspase-Glo® 3/7 Assay Substrate) (Promega, G8091) solution wasadded so as to achieve 1:1 with the medium. The operations hereafterwere carried out while being shaded. A shaker was used to mix thecontent well for two minutes at about 120 minutes⁻¹ to then be left for40 minutes at room temperature. After being left, 80 μl was moved toAssay plate (Corning, 3912, Assay plate 96 well, white polystyrene) andabsorbance was measured using GloMax-Multi Detection System (Promega,E7051).

The following mTOR inhibitors were used in this Example.

-   -   Rapamycin (Wako, #53123-88-9)    -   Everolimus (Cayman Chemical, #11597)    -   Temsirolimus (Tocris Bioscience, #5264)    -   PI-103 (Cayman Chemical, #10009209)    -   CC-223 (Cayman Chemical, #19917)    -   INK128 (Cayman Chemical, #11811)    -   AZD8055 (Cayman Chemical, #16978)    -   KU 0063794 (Tocris Bioscience, #3725)

(Results)

(Rapamycin)

The results are shown in FIG. 10. Caspase-Glo® 3/7 Assay can measure theactivity of caspase 3/7 that is involved in apoptosis induction. Thehigher the activity of caspase 3/7, the more cell damage is induced.From FIG. 10, a significant difference was not found in the activity ofcaspase 3/7 compared to control when 0.00001, 0.0001, 0.001 and 0.01 nMof rapamycin was supplemented. On the other hand, the activity ofcaspase 3/7 was found to be significantly suppressed compared to thecontrol group when 0.1, 1, 10 and 100 nM of rapamycin was supplemented.It was revealed that even with a concentration that is extremely low as0.1 nM, rapamycin significantly suppresses caspase 3/7 activity.

(Everolimus)

The results are shown in FIG. 11. The activity of caspase 3/7 was foundto be significantly suppressed compared to the control when 0.0001,0.001, 0.01 μM and 0.1 μM of everolimus was supplemented. It wasrevealed that even with a concentration that is extremely low as 0.0001μM, everolimus significantly suppresses caspase 3/7 activity.

(Temsirolimus)

The results are shown in FIG. 12. A significant difference was not foundin the activity of caspase 3/7 compared to the control when 0.000001 μMof temsirolimus was supplemented. On the other hand, the activity ofcaspase 3/7 was found to be significantly suppressed compared to thecontrol when 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 μM oftemsirolimus was supplemented. It was revealed that even with aconcentration that is extremely low as 0.00001 μM, everolimussignificantly suppresses caspase 3/7 activity.

(PI-103)

The results are shown in FIG. 13. A significant difference was not foundin the activity of caspase 3/7 compared to the control when 1 μM ofPI-103 was supplemented. On the other hand, the activity of caspase 3/7was found to be significantly suppressed compared to the control when0.001, 0.01, and 0.1 μM of PI-103 was supplemented.

(CC-223)

The results are shown in FIG. 14. The activity of caspase 3/7 was foundto be significantly suppressed compared to the control when 0.001, 0.01,0.1 and 1 μM of CC-223 was supplemented.

(INK128)

The results are shown in FIG. 15. The activity of caspase 3/7 was foundto be significantly suppressed compared to the control when 0.001, 0.01,0.1 and 1 μM of INK128 was supplemented.

(AZD8055)

The results are shown in FIG. 16. The activity of caspase 3/7 was foundto be significantly suppressed compared to the control when 0.01, 0.1and 1 μM of AZD8055 was supplemented.

(KU 0063794)

The results are shown in FIG. 17. A significant difference was not foundin the activity of caspase 3/7 compared to the control when 0.001 and0.01 μM of KU 0063794 was supplemented. On the other hand, the activityof caspase 3/7 was found to be significantly suppressed compared to thecontrol when 0.1 and 1 μM of KU 0063794 was supplemented.

Example 11: In Vivo Evaluation in a Mouse Model

This Example demonstrated the in vivo effect of an mTOR inhibitor in anevaluation system using a Fuchs' endothelial corneal dystrophy modelmouse.

Specifically, this Example instilled an mTOR inhibitor into a mousehaving type 8 collagen (Col8a2 Q455K/Q455K), which is a Fuchs'endothelial corneal dystrophy model mouse, to confirm the in vivoeffect.

(Materials and Methods)

In vivo evaluation was carried out using Alpha2 Collagen VIII (Col8a2)Q455K knock-in mouse (Hum Mol Genet. 2012 Jan. 15; 21(2): 384-93.),which is a Fuchs' endothelial corneal dystrophy model mouse. Depositionof an extracellular matrix (collagen, fibronectin, or the like) to anendothelial corneal basement membrane (Descemet's membrane) calledguttae and cell density reduction due to corneal endothelial damage werefound in this model mouse in the same manner as those found in Fuchs'endothelial corneal dystrophy in a human. Thus, this model mouse isunderstood to be a good model of Fuchs' endothelial corneal dystrophy.

It is possible to treat or prevent outbreak of Fuchs' endothelialcorneal dystrophy or delay the progression of the pathology byinhibiting the mTOR pathway of the corneal endothelium by carrying outinstillation administration, anterior chamber administration,intravitreal administration, subconjunctival administration, or systemicadministration of an mTOR inhibitor, or carrying out gene therapy tosuch a mouse. Thus, this Example used such a mouse to confirm theeffect.

(mTOR Inhibitor Eye Drop Preparation)

25 mg of Torisel® intravenous drip infusion liquid (Pfizer Inc.)(temsirolimus) was used as an eye drop of an mTOR inhibitor. Thisproduct and the liquid for dilution attached to this product are mixedin the ratio of 2:3 to prepare 92.78 μl of a 9.7 mM mixture.Furthermore, the 9.7 mM mixture was diluted with 807.22 μl of OTSUKANORMAL SALINE (Otsuka Pharmaceutical Co., Ltd.) to prepare 900 μl of a 1mM mixture in a 1.5 ml tube. Next, 900 μl of a 10 μM mixture wasprepared using 9 μl of the prepared 1 mM mixture and 891 μl of theOTSUKA NORMAL SALINE. After the preparation, the 1.5 ml tube was coveredwith aluminum foil to render a shaded state thereto, then be stored in arefrigerator at 4° C.

(Instillation Test on a Mouse)

A mouse having type 8 collagen (Col8a2^(Q455K/Q455K)), which is a Fuchs'endothelial corneal dystrophy (FECD) model mouse, was used (obtainedfrom Johns Hopkins University). Severity of FECD was graded from acorneal endothelial image of before the instillation to use FECD modelmice that are 20 to 24 weeks old with the same degree of condition. 2 μlof the prepared mTOR inhibitor eye drop (1 mM, 10 μM) was instilled intoeach of the left and right eyes of 45 mice twice a day in the morningand in the evening. OTSUKA NORMAL SALINE was used for a control. Theinstillation period was 3 months, during which the person in charge ofthe experimentation carried out the experimentation in a blinded stateregarding the mTOR inhibitor eye drop and the control eye drop (OTSUKANORMAL SALINE).

(Evaluation of the Effectiveness of the Eye Drop)

Before starting the instillation test, corneal endothelial images wereobserved with a contact corneal endothelial specular (KSSP slit-scanningwide-field contact specular microscope (Konan medical Inc., Hyogo,Japan)) for grading. After starting the instillation test, the cornealendothelial images of the mice were observed once every 4 weeks with thecontact corneal endothelial specular to evaluate the effectiveness ofthe mTOR inhibitor eye drop.

(Results)

FIG. 19 shows a typical example of a corneal endothelial cell imageobserved by the contact corneal endothelial specular in an FECD modelmouse to which 2 μl of the mTOR inhibitor eye drop (1 mM) was instilledinto each of the left and right eyes twice a day in the morning and inthe evening for 2 months. A corneal endothelial cell image in an FECDmodel mouse to which a physiological saline solution was instilled isshown as a control. Compared to the control, the size of the cornealendothelial cells are smaller and the cell density is high in anindividual into which the mTOR inhibitor eye drop was instilled.Furthermore, generation of a verrucose deposition image of anextracellular matrix called guttae that can be observed in black by thecontact corneal endothelial specular was suppressed (FIG. 19).

Corneal endothelial cell density of the control was an average of 1558cells/mm², whereas the average was 1957 cells/mm² in the mTOR inhibitoreye drop group, which was significantly higher, thereby suppressing thecorneal endothelial cell density reduction found in an FECD model mouse(FIG. 20). This suggests that generation of edema of the corneal stroma,corneal epithelial edema, or the like, which is often found when thecorneal endothelial cell density is generally about 1000 cells/mm² orlower, can be prevented by suppressing corneal endothelial cellreduction by instillation administration of the mTOR inhibitor in anFECD patient.

In addition, regarding the range of guttae, the control was an averageof 3.02%, whereas the mTOR inhibitor instillation group was the averageof 0.58%, which was significantly lower, thereby suppressing thegeneration of guttae found in an FECD model mouse (FIG. 21). When thesame analysis was carried out for 15 mice, statistically significantdifferences were found regarding the Guttae range. Guttae is known tocause decrease of visual function due to irregular reflection of lightor the like even in an FECD patient in the early stage who does not havecorneal stroma or corneal epithelial edema. Thus, this result suggeststhat decrease of visual function due to irregular reflection of lightcan be suppressed by suppressing generation of guttae by instillationadministration of an mTOR inhibitor in an FECD patient.

Example 12: Diagnosis and Therapy Example

The present invention is used when diagnosed with Fuchs' endothelialcorneal dystrophy or a similar corneal endothelial disease (specificexamples thereof include 1) observation of guttae formation, hypertrophyof the Descemet's membrane, corneal epithelial edema, or edema of thecorneal stroma by slit-lamp microscopy, 2) observation of images ofguttae or corneal endothelial disorder with a specular microscope, 3)observation of corneal edema with a Pentacam, OCT, ultrasonic cornealthickness measuring apparatus, or the like, and 4) when determined ashigh risk by genetic diagnosis). The present invention can be used intherapy as eye drops, injection into the anterior chamber,administration using controlled-release agent, intravitreal injection,subconjunctival injection, and the like.

A commercially available substance that is compatible with the JapanesePharmacopoeia, an equivalent product thereof or the like can be used aseach component other than the active ingredient.

Example 13: Preparation Example for Eye Drops

As a formulation example, this Example manufactures an eye dropcontaining an mTOR inhibitor as follows.

The composition of test substances at each concentration is shown below.

Rapamycin Effective amount (e.g., final concentration 0.1 mM) Sodiumchloride  0.85 g Sodium dihydrogen phosphate dehydrate  0.1 g(Optionally) Benzalkonium chloride 0.005 g Sodium hydroxide Optimal dosePurified water Optimal dose Total amount   100 mL (pH 7.0)

The concentration may be diluted using a base consisting of thefollowing components.

Sodium chloride  0.85 g Sodium dihydrogen phosphate dehydrate  0.1 g(Optionally) Benzalkonium chloride 0.005 g Sodium hydroxide Optimal dosePurified water Optimal dose Total amount   100 mL (pH 7.0)

Example 14: Instillation Test on a Mouse

Instillation of an mTOR inhibitor was carried out to a mouse having type8 collagen (Col8a2 Q455K/Q455K), which is a Fuchs' endothelial cornealdystrophy model mouse, in this Example. This model mouse showsdeposition of an extracellular matrix (collagen, fibronectin, or thelike), which is recognized in Fuchs' endothelial corneal dystrophy.

(Materials and Methods)

As disclosed above, the present invention is exemplified by the use ofits preferred embodiments. However, it is understood that the scope ofthe present invention should be interpreted solely based on the Claims.It is also understood that any patent, any patent application, and anyreferences cited herein should be incorporated herein by reference inthe same manner as the contents are specifically described herein. Thepresent application claims priority to Japanese Patent Application2017-118619 (filed on Jun. 16, 2017). The entire content thereof isincorporated herein by reference.

INDUSTRIAL APPLICABILITY

The present invention provides a medicament for use in treating orpreventing a corneal endothelial disorder due to transforming growthfactor-β (TGF-β), and/or overexpression of extracellular matrix incorneal endothelial cells, especially a medicament for use in treatingor preventing a corneal endothelial disorder in Fuchs' endothelialcorneal dystrophy. The present invention provides a technique availableto industries (pharmaceutical or the like) involved in techniquesassociated with formulation or the like based on such a technique.

[Sequence Listing Free Text]

SEQ ID NO: 1: Sense strand of mTOR siRNA

SEQ ID NO: 2: Antisense strand of mTOR siRNA

The invention claimed is:
 1. A method for preventing or treating an eyecondition, disorder, or disease in a subject, wherein the methodcomprises administering an effective amount of an mTOR inhibitor to thesubject, wherein the eye condition, disorder, or disease is a cornealendothelial condition, disorder, or disease due to a transforming growthfactor-β (TGF-β).
 2. The method of claim 1, wherein the cornealendothelial condition, disorder, or disease is selected from the groupconsisting of Fuchs' endothelial corneal dystrophy, post-cornealtransplant disorder, corneal endotheliitis, trauma, ophthalmic surgery,post-ophthalmic laser surgery disorder, aging, posterior polymorphousdystrophy (PPD), congenital hereditary endothelial dystrophy (CHED),idiopathic corneal endothelial disorder, and cytomegalovirus cornealendotheliitis.
 3. The method of claim 1, wherein the corneal endothelialcondition, disorder, or disease is due to overexpression ofextracellular matrix (ECM).
 4. The method of claim 3, wherein thecorneal endothelial condition, disorder, or disease is selected from thegroup consisting of Fuchs' endothelial corneal dystrophy, guttaeformation, hypertrophy of a Descemet's membrane, hypertrophy of acornea, turbidity, scar, corneal nebula, corneal macula, leucoma,photophobia, and blurred vision.
 5. The method of claim 1, wherein thecondition, disorder, or disease comprises Fuchs' endothelial cornealdystrophy.
 6. The method of claim 1, wherein the mTOR inhibitor isselected from the group consisting of rapamycin, temsirolimus,everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalisib,Ridaforolimus, NVP-BEZ235, CZ415, Torkinib, Torin 1, Omipalisib,OSI-027, PF-04691502, Apitolisib, WYE-354, Vistusertib, Torin 2,Tacrolimus, GSK1059615, Gedatolisib, WYE-125132, BGT226, Palomid 529,PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388,Zotarolimus, and Chrysophanic Acid.
 7. The method of claim 1, whereinthe mTOR inhibitor is selected from the group consisting of rapamycin,temsirolimus, and everolimus.
 8. The method of claim 1, wherein the mTORinhibitor is administered as an eye drop.
 9. The method of claim 1,wherein the mTOR inhibitor is rapamycin and is administered as an eyedrop at least about 0.1 nM.
 10. The method of claim 1, wherein the mTORinhibitor is administered as an eye drop, wherein the mTOR inhibitor israpamycin and is present in the eye drop at least about 0.1 mM.
 11. Themethod of claim 1, wherein the mTOR inhibitor is temsirolimus and isadministered as an eye drop at least about 0.01 nM.
 12. The method ofclaim 1, wherein the mTOR inhibitor is administered as an eye drop,wherein the mTOR inhibitor is temsirolimus and is present in the eyedrop at least about 0.01 mM.
 13. The method of claim 1, wherein the mTORinhibitor is everolimus and is administered as an eye drop at leastabout 0.1 nM.
 14. The method of claim 1, wherein the mTOR inhibitor isadministered as an eye drop, wherein the mTOR inhibitor is everolimusand is present in the eye drop at least about 0.1 mM.
 15. A method forpreventing or treating an eye condition, disorder, or disease in asubject, wherein the method consists essentially of administering aneffective amount of an mTOR inhibitor to the subject, wherein the eyecondition, disorder, or disease is a corneal endothelial condition,disorder, or disease due to a transforming growth factor-β (TGF-β). 16.The method of claim 15, wherein the mTOR inhibitor is rapamycin.